U.S. patent application number 16/621318 was filed with the patent office on 2020-04-16 for compositions and methods for treating cancers with covalent inhibitors of cyclin-dependent kinase 7 (cdk7).
The applicant listed for this patent is Syros Pharmaceuticals, Inc.. Invention is credited to Emmanuelle Di Tomaso, Graeme Hodgson, Shanhu Hu, Liv Helena Johannessen, Nisha Rajagopal.
Application Number | 20200113902 16/621318 |
Document ID | / |
Family ID | 64659348 |
Filed Date | 2020-04-16 |
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United States Patent
Application |
20200113902 |
Kind Code |
A1 |
Rajagopal; Nisha ; et
al. |
April 16, 2020 |
COMPOSITIONS AND METHODS FOR TREATING CANCERS WITH COVALENT
INHIBITORS OF CYCLIN-DEPENDENT KINASE 7 (CDK7)
Abstract
The present invention relates to methods of identifying subjects
suffering from various types of cancer who are more likely to
respond to treatment with a covalent CDK7 inhibitor, such as
N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-1-methylcycl-
ohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)picolinamide
(Compound 1), either alone or in combination with other classes of
anti-cancer therapies based on the presence or absence of certain
biomarkers. In addition, the present invention relates to
combinations of Compound 1 and one or more other anti-cancer
therapies, kits containing them, and the use of such combinations
in treating subjects suffering from various types of cancers.
Inventors: |
Rajagopal; Nisha; (Boston,
MA) ; Hodgson; Graeme; (Boxborough, MA) ; Di
Tomaso; Emmanuelle; (Lexington, MA) ; Johannessen;
Liv Helena; (Cambridge, MA) ; Hu; Shanhu;
(Medford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Syros Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
64659348 |
Appl. No.: |
16/621318 |
Filed: |
June 12, 2018 |
PCT Filed: |
June 12, 2018 |
PCT NO: |
PCT/US2018/037147 |
371 Date: |
December 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62518429 |
Jun 12, 2017 |
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62539912 |
Aug 1, 2017 |
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62578157 |
Oct 27, 2017 |
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62593734 |
Dec 1, 2017 |
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62641638 |
Mar 12, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C07D 401/14 20130101; A61K 31/506 20130101; A61P 35/00 20180101;
A61K 45/06 20130101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; C12Q 1/6886 20060101 C12Q001/6886; A61P 35/00 20060101
A61P035/00 |
Claims
1. A therapeutic method comprising administering a compound of the
formula ##STR00010## or a pharmaceutically acceptable salt thereof,
to a patient who has cancer and who is identified as: (a) having a
level of B-cell lymphoma-extra large (BCLXL) mRNA in the cancer
equal to or below a pre-determined threshold; and/or (b) having in
at least one of the genes involved in the RB-E2F pathway an
alteration in the DNA, an epigenetic alteration, or an alteration
in the level of expression of mRNA or protein; and/or (c) being
treated with a platinum-based therapeutic agent or whose cancer has
developed resistance to a platinum-based therapeutic agent; and/or
(d) having become or at risk of becoming resistant to treatment
with a CDK4/6 inhibitor when used alone or in combination with one
or more of an aromatase inhibitor, a selective estrogen receptor
modulator or a selective estrogen receptor degrader.
2. The therapeutic method of claim 1, wherein the cancer is a
triple negative breast cancer (TNBC), ovarian cancer, non-small
cell lung cancer, or acute myeloid leukemia (AML) and the patient
has been selected by virtue of having a level of BCLXL mRNA in the
cancer equal to or below the pre-determined threshold level.
3. The therapeutic method of claim 2, wherein the patient has
undergone, is presently undergoing, or is intending to undergo
treatment with a Bcl-2 inhibitor, such as venetoclax.
4. The therapeutic method of claim 1, wherein the patient is
selected by virtue of having one or more of: (a) a level of CCNE1
gene copy number, mRNA or protein in the cancer equal to or above a
pre-determined threshold; (b) a level of RB1 gene copy number, mRNA
or protein in the cancer equal to or below a pre-determined
threshold, or an absence of an expressed wild-type RB1 gene; (c) a
level of CDK6 mRNA equal to or above a pre-determined threshold
level; (d) a level of CCND2 mRNA equal to or above a pre-determined
threshold level; or (e) a level of CDKN2A mRNA equal to or below a
pre-determined threshold level.
5. The therapeutic method of claim 4, wherein the patient is
selected by virtue of having a level of CCNE1 gene copy number,
mRNA or protein in the cancer equal to or above a pre-determined
threshold; a level of RB1 gene copy number, mRNA or protein in the
cancer equal to or below a pre-determined threshold; or an absence
of an expressed wild-type RB1 gene.
6. The therapeutic method of claim 4, wherein the patient is
suffering from ovarian cancer, breast cancer, triple-negative
breast cancer, or hormone receptor-positive breast cancer.
7. The therapeutic method of claim 6, wherein the patient has
undergone, is presently undergoing, or is intending to undergo
treatment with a selective estrogen receptor modulator such as
tamoxifen, a selective estrogen receptor degrader such as
fulvestrant, and/or a PARP inhibitor, such as olaparib or
niraparib.
8. The therapeutic method of claim 1, wherein the patient has
become resistant to the platinum-based therapeutic agent.
9. The therapeutic method of claim 1, wherein the platinum-based
therapeutic agent is carboplatin or oxaliplatin.
10. The therapeutic method of claim 8, wherein the cancer is
ovarian cancer.
11. The therapeutic method of claim 1, wherein the patient has
undergone, is presently undergoing, or is intending to undergo
treatment with a selective estrogen receptor modulator such as
tamoxifen, or a selective estrogen receptor degrader such as
fulvestrant.
12. A therapeutic method comprising administering an effective
amount of Compound 1 ##STR00011## or a pharmaceutically acceptable
salt thereof, in a combination therapy with an effective amount of
a second agent in treating to a patient who has cancer, wherein:
(a) the cancer is TNBC, an estrogen receptor-positive (ER.sup.+)
breast cancer, pancreatic cancer, or a squamous cell cancer of the
head or neck and the second agent is a CDK4/6 inhibitor; (b) the
cancer is a breast cancer, or an ovarian cancer and the second
agent is a PARP inhibitor; (c) the cancer is AML, and the second
agent is a FLT3 inhibitor; (d) the cancer is an ovarian cancer and
the second agent is a platinum-based anti-cancer agent; (e) the
cancer is TNBC, AML, Ewing's sarcoma, or an osteosarcoma and the
second agent is a BET inhibitor; or (f) the cancer is TNBC, AML, an
ovarian cancer, or non-small cell lung cancer and the second agent
is a Bcl-2 inhibitor.
13. The therapeutic method of claim 12, wherein the cancer is AML
and the second agent is a Bcl-2 inhibitor, such as venetoclax.
14. The therapeutic method of claim 12, wherein the cancer is an
epithelial ovarian cancer, a fallopian tube cancer, a primary
peritoneal cancer, a triple negative breast cancer or a
Her2.sup.+/ER.sup.-/PR.sup.- breast cancer and the second agent is
a PARP inhibitor, such as olaparib or niraparib.
15. The therapeutic method of claim 12, wherein the cancer is an
ovarian cancer and the second agent is a platinum-based anti-cancer
agent, such as carboplatin or oxaliplatin.
16. A pharmaceutical composition comprising: (a) an effective
amount of Compound 1 ##STR00012## or a pharmaceutically acceptable
salt thereof; (b) an effective amount of a second agent selected
from a Bcl-2 inhibitor such as venetoclax, a PARP inhibitor such as
olaparib or niraparib, a platinum-based anti-cancer agent such as
carboplatin or oxaliplatin, a taxane such as paclitaxel, a CDK4/6
inhibitor such as palbociclib, ribociclib, abemaciclib, or
trilaciclib, a selective estrogen receptor modulator such as
tamoxifen, and a selective estrogen receptor degrader such as
fulvestrant; and (c) a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a U.S. National Phase Application under
35 U.S.C. .sctn. 371 of International Application No.
PCT/US2018/037147, filed Jun. 12, 2018, which claims the benefit of
the filing dates of U.S. provisional application No. 62/641,638,
filed Mar. 12, 2018, U.S. provisional application No. 62/593,734,
filed Dec. 1, 2017, U.S. provisional application No. 62/578,157,
filed Oct. 27, 2017, U.S. provisional application No. 62/539,912,
filed Aug. 1, 2017, and U.S. provisional application No.
62/518,429, filed Jun. 12, 2017. The entire content of each of
these applications is hereby incorporated by reference herein in
its entirety.
BACKGROUND OF THE INVENTION
[0002] The transcriptional kinase CDK7 (cyclin-dependent kinase 7)
has been implicated in the pathogenesis of multiple malignancies,
including leukemias (e.g., acute myeloid leukemia (AML)), breast
cancer (e.g., triple negative breast cancer (TNBC)), and ovarian
cancer, and it may play important roles in regulation of oncogenic
transcriptional dependencies and in regulation of the mitochondrial
apoptosis machinery in tumors. For example, data from recent
studies have demonstrated that TNBC cells are highly dependent on
the transcriptional regulator CDK7 and suggest that the
mitochondrial apoptosis pathway is important in mediating cell
survival in CDK7-dependent cells. Further, TNBC has been shown to
have a distinct epigenetic and transcriptional program, with
super-enhancers (SEs) mediating the expression of key oncogenic
drivers such as MYC.
SUMMARY OF THE INVENTION
[0003] The present invention features, inter alia, compositions and
methods for identifying or selecting cancer patients who are likely
to respond well to treatment with a covalent CDK7 inhibitor (i.e.,
diagnostic methods) and/or methods for treating such patients with
a covalent inhibitor of CDK7, either alone or in combination with
other classes of anti-cancer therapeutics, as described further
below. The diagnostic methods include a step of identifying or
selecting a subject suffering from a cancer that is likely to
respond well to treatment with a covalent CDK7 inhibitor, such as
N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-1-methylcycl-
ohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)picolinamide
(Compound 1), or pharmaceutically acceptable salts thereof, and the
treatment methods include a step of administering a covalent CDK7
inhibitor to an identified or selected subject. Thus, methods in
which a patient is only diagnosed as being a suitable (or good)
candidate for treatment, methods in which a selected patient within
an identified subset of patients is only treated as described
herein, and methods in which a patient is both diagnosed and
treated as described herein are encompassed by the present
invention.
[0004] For ease of reading, we will not refer to both compounds
that are covalent CDK7 inhibitors (e.g., Compound 1) and
pharmaceutically acceptable salts thereof when describing each and
every composition, method, and use within the scope of the
invention. It is to be understood that where a covalent CDK7
inhibitor described herein can be used, a pharmaceutically
acceptable salt thereof may also be useful, and that determination
(determining an appropriate salt form of a compound) is well within
the ability of one of ordinary skill in the art.
[0005] The diagnostic methods that identify or select a subject for
treatment can include a step of analyzing one or more biomarkers in
a biological sample obtained from the subject by determining,
having determined, or receiving information concerning the state of
one or more specific biomarkers (e.g., the presence, absence, or
copy number of a biomarker gene in wild type or mutant form, the
association of a biomarker gene with a super-enhancer (SE) or a SE
of a certain strength, the level of expression of the biomarker
gene (as evidenced, for example, by mRNA levels) and/or the level
of expression or activity of the protein encoded by the biomarker
gene). The state of a biomarker can be assessed in terms of any one
or more of the features just listed regardless of the precise
method or context in which the biomarker is being assessed. The
state of a given biomarker may be equal to or above a
pre-determined threshold level or equal to or below a
pre-determined threshold level. In the methods of the present
invention, one can analyze a biomarker selected from MYC, CDK18,
CDK19, CCNE1, FGFR1, or certain E2F pathway members by determining,
having determined, and/or receiving information that the state of
such a biomarker is equal to or above (e.g., above) a
pre-determined threshold level. Alternatively, or in addition, one
can analyze a biomarker selected from BCLXL, CDK7, CDK9, and RB1
(or certain E2F pathway members) by determining, having determined,
and/or receiving information that the state of such biomarker is
equal to or below (e.g., below) a pre-determined threshold level.
The choice of which biomarker(s) to utilize may depend, in part, on
the particular cancer that the subject is suffering from, as well
as other factors described herein.
[0006] The compositions of the invention include pharmaceutically
acceptable compositions that include combinations of a covalent
CDK7 inhibitor, as described herein (e.g., Compound 1 or a
pharmaceutically acceptable salt thereof), and one or more other
anti-cancer therapeutics (as described herein). In keeping with
convention, in any embodiment requiring these two agents, we may
refer to the covalent CDK7 inhibitor or a pharmaceutically
acceptable salt thereof as the "first" active agent and to the
other anti-cancer therapeutic as the "second" active agent. In case
of any doubt, the first and second agents are distinct from one
another. The compositions of the invention also include kits that
include a covalent CDK7 inhibitor and instructional materials that
describe a suitable patient, methods of identifying a suitable
patient for treatment (e.g., by any one of the diagnostic
stratification methods described herein or assessment of resistance
to a previously administered anti-cancer agent), and/or
instructions for administering the covalent CDK7 inhibitor in
combination with at least one other anti-cancer therapy or
therapeutic. The kits of the invention can also include a second
anti-cancer agent, including any one or more of the second agents
described herein.
[0007] Each therapeutic method and any diagnostic method that
employs a covalent CDK7 inhibitor may also be expressed in terms of
use and vice versa. For example, the invention encompasses the use
of a compound or composition described herein for the treatment of
a disease described herein (e.g., cancer); a compound or
composition for use in diagnosing and/or treating or a disease
(e.g., cancer); and the use of the compound or composition for the
preparation of a medicament for treating a disease described herein
(e.g., cancer).
[0008] The methods of the invention that concern diagnosing and/or
treating a disease described herein (e.g., a cancer (or use of a
covalent CDK7 inhibitor for such purposes)) may specifically
exclude any one or more of the types of cancers described herein.
For example, the invention features methods of treating cancer by
administering a compound as described herein (e.g., a compound of
Formula A (e.g., Compound 1)) with the proviso that the cancer is
not a breast cancer; with the proviso that the cancer is not a
breast cancer or a leukemia; with the proviso that the cancer is
not a breast cancer, a leukemia, or an ovarian cancer; and so
forth, with exclusions selected from any of the diseases listed
herein and with the same notion of variable exclusion from lists of
elements relevant to other aspects of the invention (e.g., chemical
substituents of a compound described herein or components of kits
and pharmaceutical compositions).
[0009] In one aspect, the invention features the use of a covalent
CDK7 inhibitor described herein, e.g., Compound 1
##STR00001##
[0010] or a pharmaceutically acceptable salt thereof, in treating
cancer in a selected patient, wherein the patient is selected by
virtue of: (a) having a level of B-cell lymphoma-extra large
(BCLXL) mRNA in the cancer equal to or below a pre-determined
threshold; and/or (b) having in at least one of the genes involved
in the RB-E2F pathway an alteration in the DNA, an epigenetic
alteration, or an alteration in the level of expression of mRNA or
protein; and/or (c) being treated with a platinum-based therapeutic
agent (e.g., carboplatin or oxaliplatin) or whose cancer has
developed resistance to a platinum-based therapeutic agent (e.g.,
carboplatin or oxaliplatin); and/or (d) having become or at risk of
becoming resistant to treatment with a CDK4/6 inhibitor when used
alone or in combination with one or more of an aromatase inhibitor,
a selective estrogen receptor modulator or a selective estrogen
receptor degrader. In the context of this use, the cancer can be a
triple negative breast cancer (TNBC), ovarian cancer, non-small
cell lung cancer, or acute myeloid leukemia (AML) and the patient
has been selected by virtue of having a level of BCLXL mRNA in the
cancer equal to or below the pre-determined threshold level. The
patient can be one who has undergone, is presently undergoing, or
is intending to undergo treatment with a Bcl-2 inhibitor, such as
venetoclax. In the context of this use, the patient can be selected
by virtue of having one or more of: a) a level of CCNE1 gene copy
number, mRNA or protein in the cancer equal to or above a
pre-determined threshold; b) a level of RB1 gene copy number, mRNA
or protein in the cancer equal to or below a pre-determined
threshold, or an absence of an expressed wild-type RB1 gene; c) a
level of CDK6 mRNA equal to or above a pre-determined threshold
level; d) a level of CCND2 mRNA equal to or above a pre-determined
threshold level; or e) a level of CDKN2A mRNA equal to or below a
pre-determined threshold level. In specific embodiments, the
patient is selected by virtue of having a level of CCNE1 gene copy
number, mRNA or protein in the cancer equal to or above a
pre-determined threshold; a level of RB1 gene copy number, mRNA or
protein in the cancer equal to or below a pre-determined threshold;
or an absence of an expressed wild-type RB1 gene. In the context of
this use, the patient can be suffering from ovarian cancer, breast
cancer, TNBC, or hormone receptor-positive breast cancer, and the
patient may be one who has undergone, is presently undergoing, or
is intending to undergo treatment with a selective estrogen
receptor modulator such as tamoxifen, a selective estrogen receptor
degrader such as fulvestrant, and/or a PARP inhibitor, such as
olaparib or niraparib.
[0011] In another aspect, the invention features the use of a
covalent CDK7 inhibitor described herein, e.g., Compound 1
##STR00002##
or a pharmaceutically acceptable salt thereof, in a combination
therapy with an effective amount of a second agent in treating a
patient who has cancer, wherein: (a) the cancer is TNBC, an
estrogen receptor-positive (ER.sup.+) breast cancer, pancreatic
cancer, or a squamous cell cancer of the head or neck; and the
second agent is a CDK4/6 inhibitor; (b) the cancer is a breast
cancer, or an ovarian cancer; and the second agent is a PARP
inhibitor; (c) the cancer is AML; and the second agent is a FLT3
inhibitor; (d) the cancer is an ovarian cancer; and the second
agent is a platinum-based anti-cancer agent; (e) the cancer is
TNBC, AML, Ewing's sarcoma, or an osteosarcoma; and the second
agent is a BET inhibitor; (f) the cancer is TNBC, AML, an ovarian
cancer, or non-small cell lung cancer; and the second agent is a
Bcl-2 inhibitor. In particular embodiments, the cancer is AML and
the second agent is a Bcl-2 inhibitor, such as venetoclax; the
cancer is an epithelial ovarian cancer, a fallopian tube cancer, a
primary peritoneal cancer, a triple negative breast cancer or a
Her2.sup.+/ER.sup.-/PR.sup.- breast cancer and the second agent is
a PARP inhibitor, such as olaparib or niraparib; the cancer is an
ovarian cancer and the second agent is a platinum-based anti-cancer
agent, such as carboplatin or oxaliplatin.
[0012] In another aspect, the invention features pharmaceutical
compositions containing a covalent CDK7 inhibitor described herein,
e.g., (a) an effective amount of Compound 1
##STR00003##
or a pharmaceutically acceptable salt thereof; (b) an effective
amount of a second agent selected from a Bcl-2 inhibitor such as
venetoclax, a PARP inhibitor such as olaparib or niraparib, a
platinum-based anti-cancer agent such as carboplatin or
oxaliplatin, a taxane such as paclitaxel, a CDK4/6 inhibitor such
as palbociclib, ribociclib, abemaciclib, or trilaciclib, a
selective estrogen receptor modulator such as tamoxifen, and a
selective estrogen receptor degrader such as fulvestrant; and (c) a
pharmaceutically acceptable carrier.
[0013] In another aspect, the invention features methods of
treating a human subject having a cancer, the method comprising
administering to a subject identified as having a level of B-cell
lymphoma-extra large (BCLXL) mRNA in the cancer equal to or below a
pre-determined threshold an effective amount of
N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-1-methylcycl-
ohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)picolinamide
(Compound 1).
[0014] In one embodiment, the invention features methods of
treating cancer, the methods including a step of administering an
effective amount of a covalent CDK7 inhibitor to a subject (e.g., a
human subject) identified as having a level of B-cell
lymphoma-extra large (BCLXL) mRNA in the cancer (e.g., in a
biological sample obtained from the patient to be treated) that is
equal to or below a pre-determined threshold (i.e., a "selected
patient"). The methods can further include a step of determining
the level of BCLXL mRNA present in a sample of cancer cells from
the subject, and this is generally true for the methods of
treatment described herein; regardless of the biomarker analyzed or
the type of cancer in question, a method of treatment can either be
carried out on an identified patient without an explicit step of
analyzing the biomarker or with an explicit step in which the
biomarker is analyzed (e.g., by obtaining a biological sample from
a subject). The human subject may have been diagnosed as having a
cancer sensitive to a covalent CDK7 inhibitor responsive to the
determination, and the state of the BCLXL biomarker can be
determined in any of the additional ways described herein. The
pre-determined threshold is a cutoff value or a prevalence cutoff.
A subject who is determined to have a cancer sensitive to a
covalent CDK7 inhibitor can additionally be administered a Bcl-2
inhibitor (e.g., venetoclax (Venclexta.RTM.)), and a subject
selected as described here (through an analysis of the state of
BCLXL) can be suffering from a breast cancer, an ovarian cancer, a
lung cancer, or a hematological cancer. More specifically, the
subject can be suffering from TNBC, ovarian cancer, non-small cell
lung cancer, or AML.
[0015] In one embodiment, the invention features methods of
treating cancer, the methods including a step of administering an
effective amount of a covalent CDK7 inhibitor to a subject (e.g., a
human subject) identified as having a MYC SE, a MYC SE strength
above a pre-determined threshold, a CDK18 SE, a CDK18 SE strength
above a pre-determined threshold, an FGFR1 SE, or an FGFR1 SE
strength above a pre-determined threshold. In some embodiments, the
method further includes a step of analyzing the SE (e.g., by
determining its presence or absence and/or its strength) in a
biological sample including cancer cells from the subject. The
human subject may have been diagnosed as having a cancer sensitive
to a CDK7 inhibitor responsive to the determination. A subject
selected as described here (through an analysis of the state of
MYC, CDK8, or FGFR1) can be suffering from a breast cancer (e.g.,
TNBC). A diagnosing step can be based on the presence (or absence)
or the strength of a MYC SE or a CDK18 SE. In one embodiment, the
subject is suffering from ovarian cancer and the diagnosis is based
on the presence or absence or the strength of an FGFR1 SE.
[0016] In one embodiment, the invention features methods of
diagnosing and treating a human subject having a cancer, the method
including the steps of: (a) diagnosing whether the subject has a
cancer sensitive to a CDK7 inhibitor based on the state of a
biomarker selected from CDK7, CDK9, CDK18 and CDK19 (e.g., a level
of CDK7, CDK9, CDK18, or CDK19 mRNA) previously determined by
analyzing a sample of cancer cells from the subject; and (b)
administering an effective amount of a covalent CDK7 inhibitor to a
subject identified as having a cancer, wherein either: (i) the
state of the CDK18 or CDK19 biomarker (e.g., the CDK18 or CDK19
mRNA level) is equal to or above a pre-determined threshold, or
(ii) the state of the CDK7 or CDK9 biomarker (e.g., the CDK7 or
CDK9 mRNA level) is equal to or below a pre-determined threshold
(i.e., the "selected subject"). These methods can further include
determining the state of a CDK biomarker selected from CDK7, CDK9,
CDK18 and CDK19 in the cancer cells of the subject; determining by
an active analytical step that may include obtaining a biological
sample from a subject. The subject may have been diagnosed as
having a cancer sensitive to a CDK7 inhibitor responsive to the
determination. As in other embodiments, the covalent CDK7 inhibitor
can be a compound of Formula A (e.g., Compound 1). Where the
biomarker is CDK7, CDK9, CDK18, or CDK19, the subject may have a
lymphoma and the diagnosing step may more specifically be based on
the level of CDK7 mRNA; the subject may have a TNBC, and the
diagnosing step may more specifically be based on the level of CDK9
mRNA; the subject may have a TNBC, and the diagnosing step may more
specifically be based on the level of CDK18 mRNA; the subject may
have a TNBC or a small cell lung cancer, and the diagnosing step
may more specifically be based on the level of CDK19 mRNA.
[0017] With regard to combination therapies and pharmaceutical
compositions that include a first and a second active agent, the
invention provides methods of treating a human subject having a
cancer with a combination of a covalent CDK7 inhibitor (as
described herein) and a CDK4/6 inhibitor. The covalent CDK7
inhibitor can be Compound 1. In any of these embodiments, the
CDK4/6 inhibitor can be Ibrance.RTM. (palbociclib), Kisqali.RTM.
(ribociclib), Verzenio.RTM. (abemaciclib), trilaciclib, G1T38,
BPI-1178, or ON 123300, and the cancer to be treated can be a
breast cancer, pancreatic cancer, lung cancer, or squamous cell
cancer of the head and neck. More specifically, the cancer to be
treated can be small cell lung cancer, non-small cell lung cancer,
TNBC, an estrogen receptor-positive (ER.sup.+) breast cancer,
pancreatic cancer or squamous cell cancer of the head and neck.
Even more specifically, the CDK4/6 inhibitor can be Ibrance.RTM.
(palbociclib), Kisqali.RTM. (ribociclib), Verzenio.RTM.
(abemaciclib). Even more specifically, the cancer to be treated is
an ER.sup.+ breast cancer.
[0018] The invention provides methods of treating a human subject
having a cancer with a combination of a CDK7 inhibitor as described
herein (e.g., a compound of Formula A (e.g., Compound 1)) and a
CDK9 inhibitor (e.g., NVP2). The cancer to be treated can be a
breast cancer and, more specifically, can be a
Her2.sup.+/ER.sup.-/PR.sup.- breast cancer.
[0019] The invention provides methods of treating a human subject
having a cancer with a combination of a CDK7 inhibitor as described
herein (e.g., a compound of Formula A (e.g., Compound 1)) and a
Flt3 inhibitor (e.g., midostaurin). The cancer to be treated can be
a hematological cancer (e.g., AML).
[0020] The invention provides methods of treating a human subject
having a cancer with a combination of a CDK7 inhibitor as described
herein (e.g., a compound of Formula A (e.g., Compound 1)) and a BET
inhibitor. In this embodiment and others specifying a BET
inhibitor, the BET inhibitor can be JQ1 or a compound disclosed in
U.S. application Ser. No. 12/810,564, which is hereby incorporated
herein by reference in its entirety. In this embodiment, the cancer
to be treated can be a hematological cancer (e.g., AML) or a breast
cancer (e.g., TNBC). In other embodiments of these methods, the
cancer to be treated is Ewing's Sarcoma.
[0021] The invention provides methods of treating a human subject
having a cancer with a combination of a covalent CDK7 inhibitor as
described herein (e.g., a compound of Formula A or Compound 1) and
a Bcl-2 inhibitor. As in other embodiments of the invention, the
Bcl-2 inhibitor can be APG-1252, S55746, BP1002, APG-2575, or
venetoclax. The cancer can be breast cancer (e.g., TNBC), an
ovarian cancer, a lung cancer (e.g., NSCLC) or a hematological
cancer (e.g., AML).
[0022] The invention provides methods of treating a human subject
having a cancer with a combination of a covalent CDK7 inhibitor
(e.g., a compound of Formula A or Compound 1) and a PARP inhibitor.
As in other embodiments, the PARP inhibitor can be Zejula.RTM.
(niraparib) or Lynpraza.RTM. (olaparib). In some embodiments of
this method, the subject is suffering from a breast cancer (e.g.,
TNBC or Her2.sup.+/ER.sup.-/PR.sup.- breast cancer), an ovarian
cancer (e.g., an epithelial ovarian cancer), a fallopian tube
cancer, or a primary peritoneal cancer.
[0023] The invention provides pharmaceutical kits for treating
cancer comprising a covalent CDK7 inhibitor, which may be a
compound of Formula A (e.g., Compound 1), and, optionally, a second
therapeutic agent selected from: (a) a Bcl-2 inhibitor, (b) a CDK9
inhibitor, (c) a Flt3 inhibitor, (d) a PARP inhibitor, (e) a BET
inhibitor, or (f) a CDK4/6 inhibitor, any of which may be selected
from those disclosed herein. The kit can include optional
instructions for: (a) reconstituting (if necessary) the covalent
CDK7 inhibitor and/or the second therapeutic agent; (b)
administering each of the covalent CDK7 inhibitor and/or the second
therapeutic agent; and/or (c) a list of specific cancers for which
the kit is useful or diagnostic methods by which they may be
determined. The kit can also include any type of paraphernalia
useful in administering the active agent(s) contained therein
(e.g., tubing, syringes, needles, sterile dressings, tape, and the
like).
[0024] The invention provides pharmaceutically acceptable
combinations comprising a CDK7 inhibitor as described herein and a
second therapeutic agent selected from: (a) a Bcl-2 inhibitor, (b)
a CDK9 inhibitor, (c) a Flt3 inhibitor, (d) a PARP inhibitor, (e) a
BET inhibitor, or (f) a CDK4/6 inhibitor (many examples of which
are provided herein and can be incorporated); and a
pharmaceutically acceptable carrier.
[0025] The invention provides methods of treating a human subject
having a cancer, the method comprising: administering to a subject
identified as having in at least one of the genes involved in the
RB-E2F pathway: (1) an alteration in the DNA (e.g. gene copy
number, mutation, methylation); (2) an epigenetic alteration (e.g.
histone methylation, histone acetylation); or (3) an alteration in
the level of expression of mRNA or protein, an effective amount of
a covalent CDK7 inhibitor, as described herein. The subject is one
identified (i.e., selected) as having an alteration in the level of
mRNA expressed from at least one gene involved in the Rb-E2F
pathway. In this aspect, the subject is determined to have either a
level of mRNA of the at least one gene involved the RbE2F pathway
equal to or above a pre-determined threshold or a level of mRNA of
the at least one gene involved the RbE2F pathway equal to or below
a pre-determined threshold, prior to administering to the subject
an effective amount of a CDK7 inhibitor.
[0026] It will be readily apparent to one of ordinary skill in the
art that for those genes in the RB-E2F pathway that are activated
or overexpressed in cancer, one would select those patients that
had (1) an alteration in the DNA encoding such gene that resulted
in increased expression (e.g. elevated gene copy number, mutation
that led to increased activity, change in methylation that led to
increased expression); (2) an epigenetic alteration associated with
that gene that resulted in increased expression (e.g. histone
methylation or histone acetylation pattern that led to increased
expression); or (3) an increase in the level of expression of mRNA
or protein encoded by that gene. For those genes in the RB-E2F
pathway that are inactivated or under-expressed in cancer, one
would select from those patients that had (1) an alteration in the
DNA encoding that gene that resulted in decreased expression or
activity (e.g. reduced gene copy number, mutation that led to
decreased activity or inactivity, change in methylation that led to
decreased expression); (2) an epigenetic alteration associated with
that gene that resulted in decreased expression (e.g. histone
methylation or histone acetylation pattern that led to decreased
expression); or (3) an decrease in the level of expression of mRNA
or protein encoded by that gene.
[0027] In some aspects relating to using RB-E2F pathway genes as
biomarkers, the invention provides a method of treating a human
subject having a cancer, which comprises administering to a subject
identified as having either (a) a level of CCNE1 mRNA or protein in
the cancer equal to or above a pre-determined threshold; and/or (b)
a level of RB1 mRNA or protein in the cancer equal to or below a
pre-determined threshold, an effective amount of a CDK7 inhibitor.
In some aspects of these embodiments, the method further comprises
determining a level of RB1 and/or CCNE1 mRNA or protein present in
a sample of cancer cells from the subject. In some aspects of these
embodiments, the human subject is diagnosed as having a cancer
sensitive to a CDK7 inhibitor responsive to the determination. In
some aspects of these embodiments, the human subject is suffering
from ovarian cancer. In some aspects of these embodiments, the
human subject is suffering from a breast cancer. In some aspects of
these embodiments, the human subject is suffering from a triple
negative breast cancer (TNBC). In some aspects of these
embodiments, the human subject is suffering from a hormone-receptor
positive (HR.sup.+) breast cancer. In some aspect of these
embodiments, the CDK7 inhibitor is Compound 1. In some aspects of
these embodiments, the CDK7 inhibitor (e.g., Compound 1) is
co-administered with a PARP inhibitor. In some embodiments the CDK7
inhibitor (e.g., Compound 1) is co-administered with a SERM or a
SERD such as tamoxifen or fulvestrant.
[0028] The invention provides a method of treating a cancer in a
human subject by administering to the subject a combination of a
CDK7 inhibitor and a platinum-based standard of care anti-cancer
agent for such cancer or a taxane. In some aspects of this
embodiment, the cancer is an ovarian cancer. In some aspects of
this thirteenth embodiment, the standard of care anti-cancer agent
is a platinum-based anti-cancer agent. In some aspects of this
embodiment, the CDK7 inhibitor is Compound 1. In some aspects of
this embodiment, the platinum-based anti-cancer agent is
carboplatin. In some aspects of this embodiment, the platinum-based
anti-cancer agent is oxaliplatin. In some aspects of this
embodiment, the human subject is, has been determined to be, or has
become resistant (after some initial responsiveness) resistant to
the platinum-based anti-cancer agent when administered as either a
monotherapy or in combination with an anti-cancer agent other than
a CDK7 inhibitor. In some aspects of this embodiment, the human
subject is determined to have become resistant to the
platinum-based anti-cancer agent when administered as a monotherapy
or in combination with an anti-cancer agent other than a CDK7
inhibitor after some initial efficacy of that prior treatment. In
some aspects of this embodiment, the standard of care anti-cancer
agent is a taxane. In some aspects of this embodiment, the taxane
is paclitaxel.
[0029] The invention provides a method of enhancing or prolonging
the efficacy of a platinum-based anti-cancer agent in a human
subject suffering from a cancer, by co-administering to the subject
the platinum-based anti-cancer agent and a CDK 7 inhibitor. In some
embodiments, the cancer is an ovarian cancer. In some embodiments,
the CDK7 inhibitor is Compound 1. In some embodiments, the
platinum-based anti-cancer agent is carboplatin or oxaliplatin.
[0030] The invention provides a method of treating HR.sup.+ breast
cancer in a human subject selected on the basis of being resistant
to treatment with a CDK4/6 inhibitor comprising the step of
administering to the subject a covalent CDK 7 inhibitor (e.g., a
compound of Formula A or Compound 1). In some embodiments, prior to
administration of the CDK7 inhibitor (e.g., Compound 1), the
subject is, has been determined to be, or has become resistant
(after some initial responsiveness) to a prior treatment with a
CDK4/6 inhibitor alone or in combination with another standard of
care agent for breast cancer other than a CDK7 inhibitor, such as
an aromatase inhibitor (e.g., letrozole, anastrozole) or a SERM or
SERD such as tamoxifen or fulvestrant. In other words, the human
subject is selected for treatment with a covalent CDK7 inhibitor
(e.g., Compound 1) on the basis of being resistant to prior
treatment with a CDK4/6 inhibitor alone or in combination with
another standard of care agent for breast cancer other than a CDK7
inhibitor. In some embodiments, the covalent CDK7 inhibitor (e.g.,
Compound 1) is co-administered with another standard of care agent,
such as an aromatase inhibitor (e.g. anastrozole, exemestane, or
letrozole) or a SERM or SERD such as tamoxifen or fulvestrant, or a
second line treatment after failure on an aromatase inhibitor or
fulvestrant. In some embodiments, prior to administration of the
covalent CDK7 inhibitor (e.g., Compound 1), the subject is, has
been determined to be, or has become resistant (after some initial
responsiveness) to treatment with a CDK4/6 inhibitor alone or in
combination with another standard of care agent for breast cancer
other than a CDK7 inhibitor, such as an aromatase inhibitor (e.g.,
anastrozole, exemestane, or letrozole), or a SERM or SERD such as
tamoxifen or fulvestrant; and the covalent CDK7 inhibitor (e.g.,
Compound 1) is co-administered with a standard of care agent for
breast cancer (e.g., a second line treatment after failure of an
aromatase inhibitor or a SERM or SERD such as tamoxifen or
fulvestrant.
[0031] The invention provides a method of diagnosing and treating a
human subject having a cancer, the method comprising: (a)
diagnosing whether the subject has a cancer sensitive to a CDK7
inhibitor based on the level of FGFR1, CDK6, CCND2, or CDKNA2, or
the absence of a wild-type RB1 gene previously determined in a
sample of cancer cells from the subject; and (b) administering an
effective amount of a CDK7 inhibitor to a subject identified as
having a cancer wherein: (a) the level of FGFR1, CDK6, or CCND2A
mRNA is equal to or above a pre-determined threshold level; (b) the
level of CDKN2A mRNA is equal to or below a pre-determined
threshold level; or (c) the subject lacks the presence of a
wild-type RB1 gene. In some aspects of these embodiments, the
covalent CDK7 inhibitor is Compound 1. In some aspects of these
embodiments, the cancer is ovarian cancer.
[0032] In a related embodiment, the invention provides methods of
treating cancer in a human subject selected on the basis of the
cancer having one or more of: (a) a level of FGFR1 mRNA equal to or
above a pre-determined threshold level; (b) a level of CDK6 mRNA
equal to or above a pre-determined threshold level; (c) a level of
CCND2 mRNA equal to or above a pre-determined threshold level; (d)
a level of CDKN2A mRNA equal to or below a pre-determined threshold
level; or (e) an absence of a wild-type RB1 gene, wherein the
selected subject is administered a covalent CDK7 inhibitor. In some
embodiments, the covalent CDK7 inhibitor is Compound 1, and the
cancer is ovarian cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIGS. 1A-1C show the results of microarrays comparing the
level of MYC mRNA (FIG. 1A), MYC copy number (FIG. 1B), or the
strength of a SE associated with MYC (FIG. 1C) in various human
cancer cell lines to their sensitivity to Compound 1. The cell
lines are partitioned by both cancer type and, for breast cancer,
by cancer subtype, and for each cancer type or subtype, the cell's
response to treatment with Compound 1 is indicated as high (gray
bars) or low (black bars). SE strength was only tested in breast
cancer cell lines. FIG. 1D shows the correlation between SE
strength and MYC mRNA expression for TNBC cells that were
responsive (gray dots) and non-responsive (black dots) to Compound
1.
[0034] FIG. 2 shows the results of a microarray comparing the level
of CDK7 mRNA in various human cancer cell lines to their
sensitivity to Compound 1. The cell lines are partitioned by cancer
type and, for each cancer type, their response to treatment with
Compound 1 (high or low).
[0035] FIG. 3 shows results of a microarray comparing the level of
CDK9 mRNA in various human cancer cell lines to/with their
sensitivity to Compound 1. The cell lines are partitioned by both
cancer type and, for each cancer type, their response to treatment
with Compound 1 (high or low).
[0036] FIG. 4 shows the results of a microarray comparing the level
of CDK19 mRNA in various human cancer cell lines to/with their
sensitivity to Compound 1. The cell lines are partitioned by both
cancer type and, for each cancer type, their response to treatment
with Compound 1 (high or low).
[0037] FIGS. 5A-5B show the results of assays comparing the
presence of a super enhancer associated with CDK18 (FIG. 5A) or
CDK18 mRNA measured in a microarray (FIG. 5B) in various human
breast cancer cell lines to their sensitivity to Compound 1. The
cell lines are further partitioned by TNBC- or non-TNBC breast
cancer subtype and, for each cancer subtype, their response to
treatment with Compound 1 (high or low).
[0038] FIGS. 6A-6B show the results of a microarray comparing the
level of BCL-XL mRNA in various human cancer cell lines to their
sensitivity to Compound 1 (FIG. 6A) or staurosporine (FIG. 6B). The
cell lines are partitioned by both cancer type and, for each cancer
type, their response to treatment with Compound 1 (high or
low).
[0039] FIGS. 7A-7B show the results of a microarray comparing the
level of CDK7 mRNA in various human cancer cell lines to their
sensitivity to Compound 1 (FIG. 7A) or staurosporine (FIG. 7B). The
cell lines are partitioned by both cancer type and, for each cancer
type, their response to treatment with Compound 1 (high or
low).
[0040] FIGS. 8A-8B show the results of a microarray comparing the
level of CDK9 mRNA in various human cancer cell lines to their
sensitivity to Compound 1 (FIG. 8A) or staurosporine (FIG. 8B). The
cell lines are partitioned by both cancer type and, for each cancer
type, their response to treatment with Compound 1 (high or
low).
[0041] FIGS. 9A-9D show the results of treatment of a THP1 AML cell
line with a combination of varying amounts of JQ1 and Compound 1
(FIGS. 9A and 9B). The results of combination treatment are plotted
as a Combination Index (CI; FIG. 9C) and as an Isobologram (FIG.
9D).
[0042] FIGS. 10A-10D show the results of treating an AML3 AML cell
line with a combination of varying amounts of JQ1 and Compound 1
(FIGS. 10A and 10B). The results of combination treatment are
plotted as a Combination Index (CI; FIG. 10C) and as an Isobologram
(FIG. 10D).
[0043] FIGS. 11A-11D show the results of treating an OCI-M1 AML
cell line with a combination of varying amounts of JQ1 and Compound
1 (FIGS. 11A and 11B). The results of combination treatment are
plotted as a Combination Index (CI; FIG. 11C) and as an Isobologram
(FIG. 11D).
[0044] FIGS. 12A-12E show the results of treating an HL60 AML cell
line with a combination of varying amounts of JQ1 and Compound 1
(FIGS. 12A and 12B). The results of combination treatment are
plotted as a Combination Index (CI; FIG. 12C) and as an Isobologram
(FIG. 12D). A comparison of cell line viability versus treatment is
shown as a bar graph in FIG. 12E.
[0045] FIGS. 13A-13D show the results of treating a THP1 AML cell
line with a combination of varying amounts of venetoclax and
Compound 1 (FIGS. 13A and 13B). The results of combination
treatment are plotted as a Combination Index (CI; FIG. 13C) and as
an Isobologram (FIG. 13D).
[0046] FIGS. 14A-14C show the results of treating an AML3 AML cell
line with a combination of varying amounts of venetoclax and
Compound 1 (FIGS. 14A and 14B). The results of combination
treatment are plotted as an Isobologram (FIG. 14C).
[0047] FIGS. 15A-15E show the results of treating a HL60 AML cell
line with a combination of varying amounts of venetoclax and
Compound 1 (FIGS. 15A and 15B). The results of combination
treatment are plotted as a Combination Index (CI; FIG. 15C), as an
Isobologram (FIG. 15D). A comparison of cell line viability versus
treatment is shown as a bar graph in FIG. 15E.
[0048] FIGS. 16A-16D show the results of treating a THP1 AML cell
line with a combination of varying amounts of the Flt3 inhibitor
midostaurin and Compound 1 (FIGS. 16A and 16B). The results of
combination treatment are plotted as a Combination Index (CI; FIG.
16C) and as an Isobologram (FIG. 16D).
[0049] FIGS. 17A-17D show the results of treating an AML3 AML cell
line with a combination of varying amounts of the Flt3 inhibitor
midostaurin and Compound 1 (FIGS. 17A and 17B). The results of
combination treatment are plotted as a Combination Index (CI; FIG.
17C) and as an Isobologram (FIG. 17D).
[0050] FIGS. 18A-18D show the results of treating a MV411 AML cell
line with a combination of varying amounts of the Flt3 inhibitor
midostaurin and Compound 1 (FIGS. 18A and 18B). The results of
combination treatment are plotted as a Combination Index (CI; FIG.
18C) and as an Isobologram (FIG. 18D).
[0051] FIGS. 19A-19E show the results of treating a AU565 breast
cancer cell line with a combination of varying amounts of the CDK9
inhibitor NVP2 and Compound 1 (FIGS. 19A and 19B). The results of
combination treatment are plotted as a Combination Index (CI; FIG.
19C), as an Isobologram (FIG. 19D). A comparison of cell line
viability versus treatment is shown as a bar graph in FIG. 19E.
[0052] FIGS. 20A-20E show the results of treating a HCC38 TNBC
breast cancer cell line with a combination of varying amounts of
the PARP inhibitor niraparib and Compound 1 (FIGS. 20A and 20B).
The results of combination treatment are plotted as a Combination
Index (CI; FIG. 20C), as an Isobologram (FIG. 20D). A comparison of
cell line viability versus treatment is shown as a bar graph in
FIG. 20E.
[0053] FIGS. 21A-21E show the results of treating a AU565 breast
cancer cell line with a combination of varying amounts of the PARP
inhibitor niraparib and Compound 1 (FIGS. 21A and 21B). The results
of combination treatment are plotted as a Combination Index (CI;
FIG. 21C), as an Isobologram (FIG. 21D). A comparison of cell line
viability versus treatment is shown as a bar graph in FIG. 21E.
[0054] FIGS. 22A-22B show the results of a microarray comparing the
level of BCL2L1 (which encodes BCL-XL) mRNA in various human cancer
cell lines (FIG. 22A) and in subsets of breast cancer cell lines
(FIG. 22B) to their sensitivity to Compound 1. The cell lines in
these figures are partitioned by both cancer type or breast cancer
subtype and, for each cancer type or subtype, their response to
treatment with Compound 1 (high or low).
[0055] FIGS. 23A-23D show the effect of Compound 1 on the
expression of various BCL2 family members in breast cancer cell
lines and ovarian cancer cells at the protein level (FIGS. 23A and
23D) and mRNA level (FIGS. 23B and 23C).
[0056] FIG. 24A shows the level of BCL2 protein in four different
AML cell lines. FIG. 24B shows the effect of Compound 1 on the
level of BCL-XL and MCL1 proteins in those same four AML cell
lines.
[0057] FIG. 25 is an isobologram showing the combined effect of
Compound 1 and venetoclax on the AML cell line KG1.
[0058] FIGS. 26A-26C show the results of treatment of a T47D breast
cancer cell line with a combination of varying amounts of the
CDK4/6 inhibitor palbociclib and Compound 1 (FIG. 26A). The results
of combination treatment are plotted as a Combination Index (CI;
FIG. 26B), as an Isobologram (FIG. 26C).
[0059] FIGS. 27A-27C show the results of treatment of a T47D breast
cancer cell line with a combination of varying amounts of the
CDK4/6 inhibitor ribociclib and Compound 1 (FIG. 27A). The results
of combination treatment are plotted as a Combination Index (CI;
FIG. 27B), as an Isobologram (FIG. 27C).
[0060] FIGS. 28A-28C show the results of treatment of a T47D breast
cancer cell line with a combination of varying amounts of the
CDK4/6 inhibitor abemaciclib and Compound 1 (FIG. 28A). The results
of combination treatment are plotted as a Combination Index (CI;
FIG. 28B), as an Isobologram (FIG. 28C).
[0061] FIGS. 29A-29B show Western blots of the protein expression
levels of various biomarkers (MCL1, BCLXL, and BCL2 in FIG. 29A;
MCL1 in FIG. 29B) as compared to .beta.-actin expression levels
after treatment with different amounts of Compound 1 in TNBC cell
lines (FIG. 29A) and in a HCC70 tumor xenograft model (FIG.
29B).
[0062] FIGS. 30A-30B show that the correlation between growth rate
(GR) of various TNBC cell lines and varying amounts of Compound 1
is dependent upon BCLXL protein expression level. Growth rate of
four different cell lines is shown in FIG. 30A. Baseline BCLXL
protein expression in those four cell lines is shown in FIG.
30B.
[0063] FIG. 31 shows the effect of Compound 1 on tumor volume in a
human TNBC cell line (HCC70) xenograft.
[0064] FIGS. 32A-32D show the effect of Compound 1 on tumor volume
in four different human TNBC patient sample xenografts. Each black
line in FIGS. 32A-32D represents a different xenograft mouse. Gray
lines represent historical tumor growth in individual untreated
mice.
[0065] FIGS. 33A-33C show the mRNA expression of biomarkers (FIG.
33A--BCL2L1; FIG. 33B--CCNE1), and the CCNE1 gene copy number (FIG.
33C) in TNBC patient sample xenografts.
[0066] FIGS. 34A-34H show the effect of Compound 1 on tumor volume
in nine different human ovarian cancer patient sample xenografts.
Each gray line represents a different xenograft mouse. Black lines
represent historical tumor growth in individual untreated mice.
[0067] FIG. 35 shows CCNE1 and RB1 protein levels in the eight
ovarian cancer xenografts analyzed in FIGS. 34A-34H.
[0068] FIG. 36 is an isobologram showing the combined effect of
Compound 1 and venetoclax on the AML cell line ML-2.
[0069] FIG. 37 is an isobologram showing the combined effect of
Compound 1 and venetoclax on the AML cell line KG-1.
[0070] FIG. 38 shows the effect of no treatment, venetoclax alone
(50 mg/kg, once a day "QD"), Compound 1 alone (40 mg/kg, once a
week "QW"), or a combination of venetoclax (50 mg/kg QD) and
Compound 1 (40 mg/kg QW) on the tumor size in a KG-1 xenograft.
[0071] FIG. 39A are isobolograms showing the combined effect of
Compound 1 and carboplatin for different ovarian cancer cell lines.
FIG. 39B is a dose-response growth curve for A2780 cells treated
with varying amounts of Compound 1 and/or carboplatin.
[0072] FIGS. 40A-40B are isobolograms showing the combined effect
of Compound 1 and oxaliplatin for different ovarian cancer cell
lines.
[0073] FIGS. 41A-41B are isobolograms showing the combined effect
of Compound 1 and the PARP inhibitor olaparib for different ovarian
cancer cell lines.
[0074] FIGS. 42A-42B are isobolograms showing the combined effect
of Compound 1 and the taxane paclitaxel for different ovarian
cancer cell lines.
[0075] FIG. 43 depicts the effect of Compound 1 versus vehicle
control on the RNA expression of CHEK1, CHEK2 and RAD51 genes in
THP-1 AML cells.
[0076] FIG. 44 depicts the effect of Compound 1, or taxol versus
vehicle and untreated controls on the RNA expression of CHEK1,
CHEK2 and RAD51 genes in various breast cancer lines.
[0077] FIGS. 45A-45E depict the rank of all enhancers in ovarian
cancer patient xenograft model OV15612, showing a super-enhancer
associated with FGFR1 (FIG. 45A), as well mRNA levels (FGFR1--FIG.
45B; CDK6--FIG. 45C; CCND2--FIG. 45D) and FGFR1 protein levels in
OV15612 (FIG. 45E) and other ovarian cancer patient xenograft
models.
[0078] FIG. 46 depicts CDKN2A mRNA expression in various ovarian
cancer patient xenograft models.
[0079] FIG. 47 depicts the effect of a combination of various
amounts of Compound 1 with various amounts JQ1 on the growth of the
Ewing's Sarcoma cell line SKES.
[0080] FIGS. 48A-48B depicts the effect of a combination of various
amounts of Compound 1 with various amounts JQ1 on the growth of the
Ewing's Sarcoma cell line RDES (FIG. 48A) and an isobologram
showing the combined effect of Compound 1 and JQ1 on that cell line
(FIG. 48B).
[0081] FIGS. 49A-49B depicts the effect of a combination of various
amounts of Compound 1 with various amounts JQ1 on the growth of the
Ewing's Sarcoma cell line A674 (FIG. 49A) and an isobologram
showing the combined effect of Compound 1 and JQ1 on that cell line
(FIG. 49B).
[0082] FIGS. 50A-50B depicts the effect of a combination of various
amounts of Compound 1 with various amounts JQ1 on the growth of the
osteocarcinoma cell line Saos2 (FIG. 50) and an isobologram showing
the combined effect of Compound 1 and JQ1 on that cell line (FIG.
50).
[0083] FIG. 51 depicts the level of expression of genes related to
homologous recombination deficiency and carboplatin sensitivity in
ovarian cell line A2780 after treatment with Compound 1 for 0, 6
and 16 hours.
[0084] FIG. 52 depicts the level of expression of genes related to
homologous recombination deficiency and carboplatin sensitivity in
ovarian cell line COV318 after treatment with Compound 1 for 0, 6
and 16 hours.
[0085] FIG. 53 depicts the level of expression of genes related to
homologous recombination deficiency and carboplatin sensitivity in
ovarian cell line TOV21G after treatment with Compound 1 for 0, 6
and 16 hours.
[0086] FIG. 54 depicts the level of expression of genes related to
homologous recombination deficiency and carboplatin sensitivity in
ovarian cell line OvCar3 after treatment with Compound 1 for 0, 6
and 16 hours.
[0087] FIG. 55 depicts the effects of carboplatin alone, Compound 1
alone, and a combination of carboplatin and Compound 1 on tumor
volume in a TOV21G xenograft model
[0088] FIG. 56 depicts the effects of carboplatin alone, Compound 1
alone, and a combination of carboplatin and Compound 1 on tumor
volume in an OvCar3 xenograft model
[0089] FIG. 57 depicts the effects of carboplatin alone, Compound 1
alone, and a combination of carboplatin and Compound 1 on tumor
volume in a A2780 xenograft model
DETAILED DESCRIPTION
[0090]
N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-1-meth-
ylcyclohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)picolinamide
(Compound 1):
##STR00004##
a covalent and selective inhibitor of CDK7, was developed to
exploit dysregulated programs thought to drive SE-mediated
transcriptional-dependencies in cancers (see WO 2015/154039).
Compound 1 has previously been shown to selectively induce
apoptosis in leukemic cells relative to non-malignant cells in
vitro, and it has demonstrated anti-tumor activity in AML
xenografts.
[0091] Despite the efficacy of Compound 1, we believe that such
efficacy will be higher in subjects that have certain genetic
signatures (i.e., biomarkers in a particular state, as described
herein). Moreover, we also believe that the efficacy of Compound 1
may be enhanced when combined with other classes of
anti-oncogenic/anti-cancer therapies or vice versa.
[0092] As used herein, the term "biological sample" refers to any
sample obtained from an individual (e.g., a patient or subject or
an animal model) suffering from a disease (or, in the case of an
animal model, a simulation of that disease) to be diagnosed or
treated by the methods of this invention or from an individual
serving in the capacity of a reference or control (or whose sample
contributes to a reference standard or control population). The
biological sample can be a tissue sample, such as a tissue section
or tissue obtained by biopsy (e.g., by needle biopsy or surgical
biopsy); a cell sample obtained from, for example, the Papanicolaou
test or blood smears; a cell sample obtained by, for example,
microdissection; a bone marrow sample (e.g., a sample of either
whole bone marrow, complete cell fractions thereof, or
subpopulations of cells therein); tissue of a xenograft, or a cell
fraction consisting of cellular fragments, cellular organelles,
and/or nucleic acids that may be obtained by lysing cells and
separating the components thereof by centrifugation or otherwise.
Other examples of biological samples include saliva, blood, serum,
urine, semen, fecal matter, cerebrospinal fluid (CSF), interstitial
fluid, mucus, tears, sweat, vaginal fluid, swabs (such as buccal
swabs). A biological sample can be obtained from a solid tumor
(e.g., a tumor of the breast, ovary, lung, or any other
cancer-affected organ disclosed herein) or a hematological tumor.
For example, the biological sample from a subject suffering from a
hematological cancer (e.g., a leukemia (e.g., AML)) can be a bone
marrow aspirate, fractionated whole blood, a PBMC (peripheral blood
mononuclear cell) fraction from the subject's whole blood, or a
PBMC sample further enriched for specific blasts using various
enrichment techniques such as antibody-linked bead enrichment
protocols, fluorescent label cell sorting, or other techniques
known in the art. In some embodiments, as will be clear from the
context in which it is described or used, the term "biological
sample" refers to a preparation that is obtained by processing a
primary sample (e.g., by removing one or more components of and/or
by adding one or more agents to the primary sample). Such a
"processed sample" may comprise, for example, nucleic acids or
proteins extracted from a sample or obtained by subjecting a
primary sample to techniques such as amplification or reverse
transcription of mRNA, isolation and/or purification of certain
components, etc.
[0093] With regard to certain values, as will be clear from the
context, the terms "about" and "approximately" are used to describe
standard variation as would be understood by one of ordinary skill
in the art or a range within plus-or-minus 10% (e.g., plus-or minus
1%) of the stated value. For example, a prevalence rank in a
population of about 80% means a prevalence rank of 72-88% (e.g.,
79.2-80.8%). In case of doubt, "about X" can be "X" (e.g., about
80% can be 80%).
[0094] As used herein, the term "biomarker" refers to an entity
whose state correlates with a particular biological event so that
it is considered to be a "marker" for that event (e.g., the
presence of a particular cancer and its susceptibility to a
covalent CDK7 inhibitor). A biomarker can be analyzed at the
nucleic acid or protein level; at the nucleic acid level, one can
analyze the presence, absence, or copy number of a gene in wild
type or mutant form, its association with a super-enhancer, and/or
its level of expression (as evidenced, for example, by mRNA
levels). At the protein level, one can analyze the level of
expression and/or activity of a protein encoded by a genetic
biomarker gene. In some embodiments, a biomarker may indicate a
therapeutic outcome or likelihood thereof. Thus, a biomarker can be
predictive, prognostic, or diagnostic and is therefore useful in
methods of identifying (thereby diagnosing) or treating a patient
as described herein.
[0095] As used herein, the term "cancer" refers to a disease in
which cells exhibit relatively abnormal, uncontrolled, and/or
autonomous growth, resulting in an aberrant growth phenotype
characterized by loss of control of cell proliferation to an extent
detrimental to the patient having the disease. Intrinsic factors
(e.g., a genetic mutation) and/or extrinsic factors (e.g., exposure
to a pathogen or carcinogen) may have contributed to a patient's
cancer. Further, the cancer can be classified by the type of tissue
in which it originated (histological type) and/or by the primary
site in the body in which the cancer first developed. Based on
histological type, cancers are generally grouped into six major
categories: carcinomas; sarcomas; myelomas; leukemias; lymphomas;
and mixed types. A cancer treated as described herein may be of any
one of these types and may comprise cells that are precancerous
(e.g., benign), malignant, pre-metastatic, metastatic, and/or
non-metastatic. A patient who has a malignancy or malignant lesion
has a cancer. A relevant cancer may be characterized by a solid
tumor or by a hematologic tumor, which may also be known as a blood
cancer. In any of the embodiments of the invention in which a
patient is suffering from a blood cancer, it can be a leukemia such
as acute lymphocytic leukemia (ALL; e.g., B cell ALL or T cell
ALL), acute myelocytic leukemia (AML; e.g., B cell AML or T cell
AML), chronic myelocytic leukemia (CML; e.g., B cell CML or T cell
CML), or chronic lymphocytic leukemia (CLL; e.g., B cell CLL (e.g.,
harry cell leukemia) or T cell CLL). The blood cancer can also be a
lymphoma such as Hodgkin lymphoma (HL; e.g., B cell HL or T cell
HL), non-Hodgkin lymphoma (NHL; e.g., B cell NHL or T cell NHL),
follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic
lymphoma (CLL/SLL), mantle cell lymphoma (MCL), a marginal zone B
cell lymphoma (e.g., splenic marginal zone B cell lymphoma),
primary mediastinal B cell lymphoma, Burkitt lymphoma,
lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia),
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, or primary central nervous system (CNS) lymphoma. The B
cell NHL can be diffuse large cell lymphoma (DLCL; e.g., diffuse
large B cell lymphoma), and the T cell NHL can be precursor T
lymphoblastic lymphoma or a peripheral T cell lymphoma (PTCL). In
turn, the PTCL can be a cutaneous T cell lymphoma (CTCL) such as
mycosis fungoides or Sezary syndrome, angioimmunoblastic T cell
lymphoma, extranodal natural kill T cell lymphoma, enteropathy type
T cell lymphoma, subcutaneous anniculitis-like T cell lymphoma, or
anaplastic large cell lymphoma. We may use the term "cancer" to
refer to a tumor or malignant neoplasm (Stedman's Medical
Dictionary, 25th ed.; Hensly ed.; Williams & Wilkins:
Philadelphia, 1990). As indicated, a cancer can manifest as an
abnormal mass of tissue whose growth surpasses and is not
coordinated with the growth of a normal tissue. A malignant
neoplasm is generally poorly differentiated (anaplasia) and has
characteristically rapid growth accompanied by progressive
infiltration, invasion, and destruction of the surrounding tissue.
Furthermore, a malignant neoplasm generally has the capacity to
metastasize to distant sites.
[0096] As used herein, the term "combination therapy" is used to
refer to those situations in which a subject is exposed to two or
more therapeutic regimens (e.g., two or more therapeutic agents).
The two or more therapeutic regimens may be administered
simultaneously, sequentially, or in overlapping dosing regimens.
"Administration" of a combination therapy may involve
administration of one or more agents to a subject who is receiving
the other agent(s) in the combination. For clarity, while
combination therapy does not require that individual agents be
administered together in a single composition (or even necessarily
at the same time), administration of a single composition
containing both agents and co-incident administration are within
the meaning of "combination therapy." As noted above, the invention
features pharmaceutical kits for treating a cancer patient, and
those kits can include a covalent CDK7 inhibitor and instructions
to administer it to a cancer patient who has undergone, is
undergoing, or will undergo treatment with a second, specified
therapeutic agent (i.e., a patient who was or is considered a
candidate for treatment with the second agent). When a patient
receives the first and second agents sequentially (i.e., when a
patient has undergone or will undergo treatment with an anti-cancer
agent other than the covalent CDK7 inhibitor), the length of time
between the administrations can vary significantly (e.g., it can be
a matter of hours, days, weeks, or months) but will be such that
one of ordinary skill in the art would view the sequential
administrations as constituting a combination therapy for the
cancer for which the patient is currently being treated.
[0097] As used herein, the term "covalent," as it relates to an
inhibitor of CDK7, refers to the manner in which the inhibitor
interacts with CDK7 at a molecular level; a covalent inhibitor of
CDK7 forms a chemical bond with CDK7 in which at least one pair of
electrons is shared between an atom in the inhibitor and an atom in
CDK7. The result is inhibition of an activity of CDK7 in a way that
benefits a patient who has cancer. We use the terms "covalent
inhibitor of CDK7" and "covalent CDK7 inhibitor"
interchangeably.
[0098] As used herein, the terms "cutoff" and "cutoff value" mean a
value measured in an assay that defines the dividing line between
two subsets of a population (e.g., responders and non-responders
(e.g., responders and non-responders to a CDK7 inhibitor). Thus,
values that are equal to or higher than the cutoff value defines
one subset of the population, and values that are lower than the
cutoff value defines the other subset of the population.
[0099] As used herein, "diagnostic information" is information that
is useful in determining whether a patient has a disease and/or in
classifying (stratifying) the disease into a genotypic or
phenotypic category or any category having significance with regard
to the prognosis of the disease or its likely response to treatment
(either treatment in general or any particular treatment described
herein). Similarly, "diagnosis" refers to obtaining or providing
any type of diagnostic information, including, but not limited to,
whether a subject is likely to have or develop a disease; whether
that disease has or is likely to reach a certain state or stage or
to exhibit a particular characteristic (e.g., resistance to a
therapeutic agent); information related to the nature or
classification of a tumor; information related to prognosis (which
may also concern resistance); and/or information useful in
selecting an appropriate treatment (e.g., selecting a covalent CDK7
inhibitor for a patient identified as having a cancer that is
likely to respond to such an inhibitor or other treatment). A
patient classified (stratified) according to a method described
herein and selected for treatment with a covalent CDK7 inhibitor is
likely to respond well to the treatment, meaning that such a
patient is more likely to be successfully treated than a patient
with the same type of cancer who has not been so identified and is
not in the same strata. Available treatments include therapeutic
agents and other treatment modalities such as surgery, radiation,
etc., and selecting an appropriate treatment encompasses the choice
of withholding a particular therapeutic agent; the choice of a
dosing regimen; and the choice of employing a combination therapy.
Diagnostic information can be used to stratify patients and is thus
useful in identifying and classifying a given patient according to,
for example, biomarker status. Obtaining diagnostic information can
constitute a step in any of the patient stratification methods
described herein.
[0100] One of ordinary skill in the art will appreciate that the
term "dosage form" may be used to refer to a physically discrete
unit of an active agent (e.g., a therapeutic or diagnostic agent)
for administration to a subject. Typically, each such unit contains
a predetermined quantity of active agent. In some embodiments, such
quantity is a unit dosage amount (or a whole fraction thereof)
appropriate for administration in accordance with a dosing regimen
that has been determined to correlate with a desired or beneficial
outcome when administered to a relevant population (i.e., with a
therapeutic dosing regimen). Those of ordinary skill in the art
appreciate that the total amount of a therapeutic composition or
agent administered to a particular subject is determined by one or
more attending physicians and may involve administration of
multiple dosage forms.
[0101] One of ordinary skill in the art will appreciate that the
term "dosing regimen" may be used to refer to a set of unit doses
(typically more than one) that are administered individually to a
subject, separated by equal or unequal periods of time. A given
therapeutic agent typically has a recommended dosing regimen, which
may involve one or more doses, each of which may contain the same
unit dose amount or differing amounts. In some embodiments, a
dosing regimen comprises a first dose in a first dose amount,
followed by one or more additional doses in a second dose amount
that is different from the first dose amount. In some embodiments,
a dosing regimen is correlated with a desired or beneficial outcome
when administered across a relevant population (i.e., the regimen
is a therapeutic dosing regimen).
[0102] As used herein, an "effective amount" of an agent (e.g., a
chemical compound described herein), such as a compound of Formula
(A) or Compound 1, refers to an amount that produces or is expected
to produce the desired effect for which it is administered. The
effective amount will vary depending on factors such as the desired
biological endpoint, the pharmacokinetics of the compound
administered, the condition being treated, the mode of
administration, and characteristics of the subject, as discussed
further below and recognized in the art. The term can be applied to
therapeutic and prophylactic methods. For example, a
therapeutically effective amount is one that reduces the incidence
and/or severity of one or more signs or symptoms of the disease.
For example, in treating a cancer, an effective amount may reduce
the tumor burden, stop tumor growth, inhibit metastasis or prolong
patient survival. One of ordinary skill in the art will appreciate
that the term does not in fact require successful treatment be
achieved in any particular individual. Rather, a therapeutically
effective amount is that amount that provides a particular desired
pharmacological response in a significant number of subjects when
administered to patients in need of such treatment. In some
embodiments, reference to a therapeutically effective amount may be
a reference to an amount administered or an amount measured in one
or more specific tissues (e.g., a tissue affected by the disease)
or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.).
Effective amounts may be formulated and/or administered in a single
dose or in a plurality of doses, for example, as part of a dosing
regimen.
[0103] As used herein, an "enhancer" is a region of genomic DNA
that helps regulate the expression of genes up to 1 Mbp away. An
enhancer may overlap, but is often not composed of, gene coding
regions. An enhancer is often bound by transcription factors and
designated by specific histone marks.
[0104] As used herein, the term "patient" or "subject" are used
interchangeably and each refers to any organism to which a provided
composition is or may be administered, e.g., for experimental,
diagnostic, prophylactic, and/or therapeutic purposes. Typical
patients include animals (e.g., mammals such as mice, rats,
rabbits, dogs, cats, non-human primates, and humans). The patient
(e.g., a human) may be treated pre-natally or post-natally, and
genetically-born males or females may be subjected to a method
described herein regardless of the stage of life at which diagnosis
and/or treatment may be advised (e.g., a patient can be an infant,
child, adolescent, young adult, middle-aged adult, or senior
adult). A patient may be suffering from or susceptible to one or
more diseases, disorders, or conditions and may display one or more
signs or symptoms of a disease, disorder, or condition. We may tend
to use the term "subject" when referring to an individual subjected
to a diagnostic method or who has provided a biological sample for
reference or for analysis within a reference population. Similarly,
we may tend to use the term "patient" when referring to an
individual subjected to a therapeutic method. However, it is to be
understood that any individual, whether human or not, and whether
designated as a "patient" or a "subject" can be subjected to the
diagnostic methods described herein, the therapeutic methods
described herein, or both.
[0105] As used herein, the term "pharmaceutically acceptable salt"
refers to a salt that is, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like, and is commensurate with a reasonable
benefit/risk ratio. Pharmaceutically acceptable salts are well
known in the art (see Berge et al., J. Pharmaceutical Sciences,
66:1-19, 1977, incorporated herein by reference). Pharmaceutically
acceptable salts of the compounds described herein include those
derived from suitable inorganic and organic acids and bases.
Examples of pharmaceutically acceptable, nontoxic acid addition
salts are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid, and perchloric acid or with organic acids such as acetic
acid, oxalic acid, maleic acid, tartaric acid, citric acid,
succinic acid, or malonic acid or by using other methods known in
the art such as ion exchange. Other pharmaceutically acceptable
salts include adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, MALAT1e, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p
toluenesulfonate, undecanoate, valerate salts, and the like. Salts
derived from appropriate bases include alkali metal, alkaline earth
metal, ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4 salts.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
[0106] As used herein, the term "population" means some number of
items (e.g., at least 30, 40, 50, or more) sufficient to reasonably
reflect the distribution, in a larger group, of the value being
measured in the population. Within the context of the present
invention, the population can be a discrete group of humans,
laboratory animals, or cells lines (for example) that are
identified by at least one common characteristic for the purposes
of data collection and analysis. For example, a "population of
samples" refers to a plurality of samples that is large enough to
reasonably reflect the distribution of a value (e.g., a value
related to the state of a biomarker) in a larger group of samples.
The items in the population may be biological samples, as described
herein. For example, each sample in a population of samples may be
cells of a cell line or a biological sample obtained from a subject
or a xenograft (e.g., a tumor grown in a mouse by implanting a
tumorigenic cell line or a patient sample into the mouse). As
noted, individuals within a population can be a discrete group
identified by a common characteristic, which can be the same
disease, condition, or disorder (e.g., the same type of cancer),
whether the sample is obtained from living beings suffering from
the same type of cancer or a cell line or xenograft representing
that cancer.
[0107] The term "prevalence cutoff," as used herein in reference to
a specified value (e.g., the strength of a SE associated a
biomarker disclosed herein) means the prevalence rank that defines
the dividing line between two subsets of a population (e.g., a
subset of "responders" and a subset of "non-responders," which, as
the names imply include subjects who are likely or unlikely,
respectively, to experience a beneficial response to a therapeutic
agent or agents). Thus, a prevalence rank that is equal to or
higher (e.g., a lower percentage value) than the prevalence cutoff
defines one subset of the population; and a prevalence rank that is
lower (e.g., a higher percentage value) than the prevalence cutoff
defines the other subset of the population.
[0108] As used herein, the term "prevalence rank" for a specified
value (e.g., the mRNA level of a specific biomarker) means the
percentage of a population that are equal to or greater than that
specific value. For example, a 35% prevalence rank for the amount
of mRNA of a specific biomarker in a test cell means that 35% of
the population have that level of biomarker mRNA or greater than
the test cell.
[0109] As used herein, the terms "prognostic information" and
"predictive information" are used to refer to any information that
may be used to indicate any aspect of the course of a disease or
condition either in the absence or presence of treatment. Such
information may include, but is not limited to, the average life
expectancy of a patient, the likelihood that a patient will survive
for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.),
the likelihood that a patient will be cured of a disease, the
likelihood that a patient's disease will respond to a particular
therapy (wherein response may be defined in any of a variety of
ways). Prognostic and predictive information are included within
the broad category of diagnostic information.
[0110] As used herein, the term "rank ordering" means the ordering
of values from highest to lowest or from lowest to highest.
[0111] As used herein, the terms "Rb-E2F pathway" and "Rb-E2F
family" refer to a set of genes whose expression regulates the
activity of the RB gene family, which in turn regulates the
activity of the E2F family of transcription factors that are
required for entry into and progression through the cell cycle. The
following table contains a list of genes in the RB-E2F family, an
indication of the functions of the encoded proteins, and the status
of these biomarkers in cancer. We use the shorthand "activated or
overexpressed" to indicate that the copy number, or level of
expression of this gene is known to be higher in certain cancers as
compared to healthy subjects. In some aspects of this invention,
the pre-determined threshold for such activated or overexpressed
genes is the level (e.g., mRNA level, protein level, gene copy
number, strength of enhancer associated with the gene) that is
present in a cancer patient known to have a higher level than a
healthy subject. We use the shorthand "inactivated or
underexpressed" to indicate that the copy number, or level of
expression of this gene is known to be lower in patients having
certain cancers as compared to healthy subjects. In some aspects of
this invention, the pre-determined threshold for such inactivated
or underexpressed genes is the level (e.g., mRNA level, protein
level, gene copy number, strength of enhancer associated with the
gene) that is present in a cancer patient known to have a lower
level than a healthy subject.
TABLE-US-00001 Gene Function Status in Cancer E2F1 E2F family -
transcriptional Activated or overexpressed control of cell cycle
entry E2F2 E2F family - transcriptional Activated or overexpressed
control of cell cycle entry E2F3 E2F family - transcriptional
Activated or overexpressed control of cell cycle entry E2F4 E2F
family - transcriptional Activated or overexpressed control of cell
cycle entry E2F5 E2F family - transcriptional Activated or
overexpressed control of cell cycle entry E2F6 E2F family -
transcriptional Activated or overexpressed control of cell cycle
entry E2F7 E2F family - transcriptional Activated or overexpressed
control of cell cycle entry E2F8 E2F family - transcriptional
Activated or overexpressed control of cell cycle entry RB1 RB
family - E2F family Inactivated or underexpressed inhibition RBL1
RB family - E2F family Inactivated or underexpressed inhibition
RBL2 RB family - E2F family Inactivated or underexpressed
inhibition CDK4 RB family inhibition Activated or overexpressed
CDK6 RB family inhibition Activated or overexpressed CDK2 RB family
inhibition Activated or overexpressed CCND1 CDK4/6 regulation
Activated or overexpressed CCND2 CDK4/6 regulation Activated or
overexpressed CCND3 CDK4/6 regulation Activated or overexpressed
CDKN2A CDK4/6 regulation Inactivated or underexpressed CDKN2B
CDK4/6 regulation Inactivated or underexpressed CDKN2C CDK4/6
regulation Inactivated or underexpressed CDKN2D CDK4/6 regulation
Inactivated or underexpressed CCNE1 CDK2 regulation Activated or
overexpressed CCNE2 CDK2 regulation Activated or overexpressed
CDKN1A CDK2 regulation Inactivated or underexpressed CDKN1B CDK2
regulation Inactivated or underexpressed CDKN1C CDK2 regulation
Inactivated or underexpressed FBXW7 CCNE regulation Inactivated or
underexpressed
[0112] As used herein, a "reference" refers to a standard or
control relative to which a comparison is performed. For example,
an agent, subject (or patient), population, sample, sequence, or
value of interest is compared with a reference agent, subject (or
patient), population, sample, sequence or value. The reference can
be analyzed or determined substantially simultaneously with the
analysis or determination of the item of interest or it may
constitute a historical standard or control, determined at an
earlier point in time and optionally embodied in a tangible medium.
One of ordinary skill in the art is well trained in selecting
appropriate references, which are typically determined or
characterized under conditions that are comparable to those
encountered by the item of interest. One of ordinary skill in the
art will appreciate when sufficient similarities are present to
justify reliance on and/or comparison to a particular possible
reference as a standard or control.
[0113] As used herein, a "response" to treatment is any beneficial
alteration in a subject's condition that results from, or that
correlates with, treatment. The alteration may be stabilization of
the condition (e.g., inhibition of deterioration that would have
taken place in the absence of the treatment), amelioration of,
delay of onset of, and/or reduction in frequency of one or more
signs or symptoms of the condition, improvement in the prospects
for cure of the condition, greater survival time, and etc. A
response may be a subject's response or a tumor's response.
[0114] As used herein, when the term "strength" is used to refer to
a portion of an enhancer or a SE, it means the area under the curve
of the number of H3K27Ac or other genomic marker reads plotted
against the length of the genomic DNA segment analyzed. Thus,
"strength" is an integration of the signal resulting from measuring
the mark at a given base pair over the span of the base pairs
defining the region being chosen to measure.
[0115] As used herein, the term "super-enhancer" (SE) refers to a
subset of enhancers that contain a disproportionate share of
histone marks and/or transcriptional proteins relative to other
enhancers in a particular cell or cell type. Genes regulated by SEs
are predicted to be of high importance to the function of a cell.
SEs are typically determined by rank ordering all of the enhancers
in a cell based on strength and determining, using available
software such as ROSE (bitbucket.org/young computation/rose), the
subset of enhancers that have significantly higher strength than
the median enhancer in the cell (see, e.g., U.S. Pat. No.
9,181,580, which is hereby incorporated by reference herein in its
entirety).
[0116] As used herein, the terms "threshold" and "threshold level"
mean a level that defines the dividing line between two subsets of
a population (e.g., responders and non-responders). A threshold or
threshold level may be a prevalence cutoff or a cutoff value.
[0117] As used herein, the terms "treatment," "treat," and
"treating" (and other grammatical variants thereof) refer to
reversing, alleviating, delaying the onset of, and/or inhibiting
the progress of a "pathological condition" (e.g., a disease,
disorder, or condition, or one or more signs or symptoms thereof)
described herein. In some embodiments, "treatment," "treat," and
"treating" require that signs or symptoms of the disease disorder
or condition have developed or have been observed. In other
embodiments, treatment may be administered in the absence of signs
or symptoms of the disease or condition (e.g., in light of a
history of symptoms and/or in light of genetic or other
susceptibility factors). Treatment may also be continued after
symptoms have resolved, for example, to delay or inhibit
recurrence.
[0118] The terms "condition," "disease," and "disorder" are used
interchangeably herein unless the context clearly indicates
otherwise. Similarly, as the invention relates to compositions and
methods for diagnosing and treating patients who have cancer, the
terms "active agent," "anti-cancer agent," "pharmaceutical agent,"
and "therapeutic agent" are used interchangeably (unless the
context clearly indicates otherwise) and the compounds described
herein as covalent inhibitors of CDK7, including Compound 1 and
those conforming to Formula (A), would be understood by one of
ordinary skill in the art as active, anti-cancer, pharmaceutical,
or therapeutic agents.
[0119] The compositions and methods described herein, including
pharmaceutical kits and compositions, therapies employing one or
more active agents, and diagnostic or patient stratification
methods, can include or employ any covalent inhibitor of CDK7,
particularly covalent inhibitors of CDK7 that form a covalent bond
with the --SH group of Cys312 of CDK7 or an equivalent cysteine
residue in a mutant form of CDK7 (i.e., the same cysteine residue
but bearing a different amino acid number because of amino acid
insertions and/or deletions in such mutants). Such covalent
inhibitors of CDK7 will contain an electrophilic moiety that is
capable of reacting with the nucleophilic --SH moiety of CDK7
Cys312 to form a covalent bond between the inhibitor and
Cys312.
[0120] Covalent CDK7 inhibitors suitable for use in the
compositions and methods described herein include those conforming
to structural formula (A):
##STR00005##
and pharmaceutically acceptable salts thereof.
[0121] E is a chemical group that is moderately hydrophobic with a
fragment cLogP between 1.0 and 3.0, which associates with a
hydrophobic pocket exposed by the inactive form of CDK7
characterized by a closed conformation of the activation loop (DFG
"out"), and may optionally contain at least one hydrogen bond donor
moiety that forms a hydrogen bond to the nitrogen of CDK7 residue
lysine 41 (Lys41);
[0122] L.sup.1e is a linker group ranging from 0 to 3 atoms in
length;
[0123] R.sup.E is an electrophilic group that forms a covalent bond
with the --SH group of Cys312 of CDK7;
[0124] U is a chemical group that contains at least one hydrogen
bond acceptor moiety that forms a hydrogen bond to the backbone
amide --NH-- of CDK7 residue methionine 94 (Met94);
[0125] L.sup.X is a linker group ranging from 0 to 5 atoms in
length, which may contain at least one hydrogen bond donor moiety
that forms a hydrogen bond to the backbone amide --CO-- group of
CDK7 residue methionine 94 (Met94);
[0126] U and L.sup.X may be optionally taken together to form a
cyclic structure; and
[0127] G is a chemical group that spans a total length of
approximately 20 to approximately 30 {acute over (.ANG.)}.
[0128] In some embodiments, E, of Formula A, is an optionally
substituted heteroaryl group. More specifically, E can be an
optionally substituted heteroaryl ring of any one of the Formulae
(i-1)-(i-6):
##STR00006##
wherein:
[0129] each instance of V.sup.1, V.sup.2, V.sup.3, V.sup.4,
V.sup.5, V.sup.6, V.sup.7, V.sup.8, V.sup.9, V.sup.10, V.sup.11,
V.sup.12, V.sup.13, V.sup.14 and V.sup.15 is independently O, S, N,
N(R.sup.A1), C, or C(R.sup.A2);
[0130] each instance of R.sup.A1 is independently selected from
hydrogen, deuterium, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, and
optionally substituted heteroaryl;
[0131] each instance of R.sup.A2 is independently selected from
hydrogen, deuterium, halogen, --CN, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, --OR.sup.A2a,
--N(R.sup.A2a).sub.2, and --SR.sup.A2a, wherein each occurrence of
R.sup.A2a is independently selected from hydrogen, optionally
substituted acyl, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, and optionally substituted heteroaryl,
or
[0132] any two R.sup.A1, any two R.sup.A2, or one R.sup.A1 and one
R.sup.A2 are joined to form an optionally substituted carbocyclic,
optionally substituted heterocyclic, optionally substituted aryl,
or optionally substituted heteroaryl ring.
[0133] In some embodiments, U and L.sup.X are not taken together to
form a heteroaryl; and U is a nitrogen-containing heteroaryl. U can
be an optionally substituted pyrimidine.
[0134] In some embodiments, G consists of two cyclic moieties bound
to one another through a 1 to 3 atom linker. In some embodiments,
the cyclic moiety in G bound to R.sup.E is an optionally
substituted aryl or heteroaryl ring. In more specific embodiments,
the cyclic moiety in G bound to R.sup.E is an optionally
substituted phenyl or pyridinyl ring. The cyclic moiety in G bound
to L.sup.X can be an optionally cycloalkyl or saturated
heterocyclyl ring. The cyclic moiety in G bound to L.sup.X can be
an optionally substituted cyclohexyl ring.
[0135] In some embodiments, R.sup.E is any one of the Formulae
(ii-1)-(ii-19):
##STR00007## ##STR00008## ##STR00009##
wherein:
[0136] L.sup.3 is a bond, an optionally substituted C.sub.1-C.sub.7
alkylene, or an optionally substituted C.sub.2-C.sub.7 alkenylene
or alkynylene, wherein one or more methylene units of the alkylene,
alkenylene or alkynylene are optionally and independently replaced
with --O--, --S--, --S(O)--, --S(O).sub.2, or --N(R.sup.6)--;
[0137] L.sup.4 is a bond, an optionally substituted C.sub.1-C.sub.4
alkylene, or an optionally substituted C.sub.2-C.sub.4 alkenylene
or alkynylene;
[0138] each of R.sup.E1, R.sup.E2 and R.sup.E3 is independently
selected from hydrogen, deuterium, halogen, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, --CH.sub.2OR.sup.9, --CH.sub.2N(R.sup.9).sub.2,
--CH.sub.2SR.sup.9, --CN, --OR.sup.9, --N(R.sup.9).sub.2, and
--SR.sup.9, wherein each occurrence of R.sup.9 is independently
selected from hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, and optionally substituted heteroaryl,
or two R.sup.9 are taken together to form an optionally substituted
heterocyclyl, or
[0139] R.sup.E1 and R.sup.E3, or R.sup.E2 and R.sup.E3, or R.sup.E1
and R.sup.E2 are joined to form an optionally substituted
carbocyclic or optionally substituted heterocyclic ring;
[0140] R.sup.E4 is a leaving group;
[0141] Y is O, S, or N(R.sup.6), wherein R.sup.6 is hydrogen, or
--C.sub.1-C.sub.6 alkyl; and
[0142] z is 0, 1, 2, 3, 4, 5, or 6.
[0143] Examples of the above-described covalent CDK7 inhibitors can
be found in PCT publications WO2014063068 (U.S. application Ser.
No. 14/436,496), WO2015058126 (U.S. application Ser. No.
15/030,249), WO2015058163 (U.S. application Ser. No. 15/030,265),
WO2015058140 (U.S. application Ser. No. 15/030,245), WO2015154039
(U.S. application Ser. No. 15/301,815), and WO2015154022 (U.S.
application Ser. No. 15/301,819), the disclosures of which are
hereby incorporated herein by reference in their entireties.
[0144] In some embodiments, the covalent CDK7 inhibitor is Compound
1 or a pharmaceutically acceptable salt thereof; in some
embodiments, the covalent CDK7 inhibitor is Compound 1.
[0145] An enhancer or SE can be identified by various methods known
in the art (see Hinsz et al., Cell, 155:934-947, 2013; McKeown et
al., Cancer Discov., 7(10):1136-53, 2017; and PCT/US2013/066957,
each of which are hereby incorporated herein by reference in their
entireties). Identifying a SE can be achieved by obtaining a
biological sample from a patient (e.g., from a biopsy or other
source, as described herein). The important metrics for enhancer
measurement occur in two dimensions: along the length of the DNA
over which genomic markers (e.g., H3K27Ac) are contiguously
detected and the compiled incidence of genomic marker at each base
pair along that span of DNA, the compiled incidence constituting
the magnitude. The measurement of the area under the curve ("AUC")
resulting from integration of length and magnitude analyses
determines the strength of the enhancer. The strength of the MYC,
CDK18, CDK19, CCNE1, or FGFR1 SEs relative to an appropriate
reference can be used to diagnose (stratify) a patient and thereby
determine whether a subject is likely to respond well to Compound 1
or another of the covalent CDK7 inhibitors described herein. It
will be readily apparent to one of ordinary skill in the art,
particularly in view of the instant specification, that if the
length of DNA over which the genomic markers is detected is the
same for each of MYC, CDK18, CDK19, CCNE1, or FGFR1 and the
reference/control, then the ratio of the magnitude of the MYC,
CDK18, CDK19, CCNE1, or FGFR1 SE relative to the control will be
equivalent to the strength and may also be used to determine
whether a subject will be responsive to a covalent CDK7 inhibitor
(such as Compound 1 or another compound described herein). The
strength of the MYC, CDK18, CDK19, CCNE1, or FGFR1 SE in a cell can
be normalized before comparing it to other samples. Normalization
is achieved by comparison to a region in the same cell known to
comprise a ubiquitous SE or enhancer that is present at similar
levels in all cells. One example of such a ubiquitous
super-enhancer region is the MALAT1 super-enhancer locus
(chr11:65263724-65266724) (genome build hg19).
[0146] It has been determined through H3K27Ac ChIP-seq
(ChIP-sequencing) methods that there is a SE locus associated with
the MYC gene at chr8:128628088-128778308; a SE locus associated
with the CDK18 gene at chr1:205399084-205515396; a SE locus
associated with the CDK19 gene at chr6:110803523-110896277; a SE
locus associated with the CCNE1 gene at chr19:30418503-30441450;
and a SE locus associated with the FGFR1 gene at
chr8:38233326-38595483. All loci are based on the Gencode v19
annotation of the human genome build hg19/GRCh37.
[0147] ChIP-seq is used to analyze protein interactions with DNA by
combining chromatin immunoprecipitation (ChIP) with massively
parallel DNA sequencing to identify the binding sites of
DNA-associated proteins. It can be used to map global binding sites
precisely for any protein of interest. Previously, ChIP-on-chip was
the most common technique utilized to study these protein--DNA
relations. Successful ChIP-seq is dependent on many factors
including sonication strength and method, buffer compositions,
antibody quality, and cell number (see, e.g., Furey, Nature Reviews
Genetics 13:840-852, 2012); Metzker, Nature Reviews Genetics
11:31-46, 2010; and Park, Nature Reviews Genetics 10:669-680,
2009). Genomic markers other than H3K27Ac that can be used to
identify SEs using ChIP-seq include P300, CBP, BRD2, BRD3, BRD4,
components of the mediator complex (Loven et al., Cell,
153(2):320-334, 2013), histone 3 lysine 4 monomethylated (H3K4me1),
and other tissue-specific enhancer tied transcription factors
(Smith and Shilatifard, Nature Struct. Mol. Biol., 21(3):210-219,
2014; and Pott and Lieb, Nature Genetics, 47(1):8-12, 2015).
Quantification of enhancer strength and identification of SEs can
be determined using SE scores (McKeown et al., Cancer Discov.
7(10):1136-1153, 2017; DOI: 10.1158/2159-8290.CD-17-0399).
[0148] In some instances, H3K27Ac or other marker ChIP-seq data SE
maps of the entire genome of a cell line or a patient sample
already exist. One would then simply determine whether the strength
or ordinal rank of the enhancer or SE in such maps at the
chr8:128628088-128778308 (genome build hg19) locus was equal to or
above the pre-determined threshold level. In some embodiments, one
would simply determine whether the strength, or ordinal rank of the
enhancer or super-enhancer in such maps at the
chr1:205399084-205515396 (genome build hg19) locus was equal to or
above the pre-determined threshold level.
[0149] It should be understood that the specific chromosomal
location of MYC, CDK18, CDK19, CCNE1, or FGFR1 and MALAT1 may
differ for different genome builds and/or for different cell types.
However, one of ordinary skill in the art, particularly in view of
the instant specification, can determine such different locations
by locating in such other genome builds specific sequences
corresponding to the MYC, CDK18, CDK19, CCNE1, or FGFR1 and/or
MALAT1 loci in genome build hg 19.
[0150] Other methods that can be used to identify SEs in the
context of the present methods include chromatin
immunoprecipitation (Delmore et al., Cell, 146(6)904-917, 2011),
chip array (ChIP-chip), and chromatin immunoprecipitation followed
by qPCR (ChIP-qPCR) using the same immunoprecipitated genomic
markers and oligonucleotide sequences that hybridize to the
chr8:128628088-128778308 (genome build hg19) MYC locus or
chr1:205399084-205515396 (genome build hg19) CDK18 locus (for
example). In the case of ChIP-chip, the signal is typically
detected by intensity fluorescence resulting from hybridization of
a probe and input assay sample as with other array-based
technologies. For ChIP-qPCR, a dye that becomes fluorescent after
intercalating the double stranded DNA generated in the PCR reaction
is used to measure amplification of the template.
[0151] In some embodiments, determination of whether a cell has a
MYC, CDK18, CDK19, CCNE1, or FGFR1 SE strength equal to or above a
requisite threshold level is achieved by comparing MYC, CDK18,
CDK19, CCNE1, or FGFR1 enhancer strength in a test cell to the
corresponding MYC, CDK18, CDK19, CCNE1, or FGFR1 strength in a
population of cell samples, wherein each of the cell samples is
obtained from a different source (e.g., a different subject, a
different cell line, a different xenograft) reflecting the same
disease to be treated. In some embodiments, only primary tumor cell
samples from subjects are used to determine the threshold level. In
some aspects of these embodiments, at least some of the samples in
the population will have been tested for responsiveness to a
specific CDK7 inhibitor (e.g., Compound 1) to establish: (a) the
lowest MYC, CDK18, CDK19, CCNE1, or FGFR1 enhancer strength of a
sample in the population that responds to that specific compound
("lowest responder"); and, optionally, (b) the highest MYC, CDK18,
CDK19, CCNE1, or FGFR1 enhancer strength of a sample in the
population that does not respond to that specific compound
("highest non-responder"). In these embodiments, a cutoff of MYC,
CDK18, CDK19, CCNE1, or FGFR1 enhancer strength above which a test
cell would be considered responsive to that specific compound is
set: i) equal to or up to 5% above the MYC, CDK18, CDK19, CCNE1, or
FGFR1 enhancer strength in the lowest responder in the population;
or ii) equal to or up to 5% above the MYC, CDK18, CDK19, CCNE1, or
FGFR1 enhancer strength in the highest non-responder in the
population; or iii) a value in between the MYC, CDK18, CDK19,
CCNE1, or FGFR1 enhancer strength of the lowest responder and the
highest non-responder in the population.
[0152] In the above embodiments, not all of the samples in a
population necessarily are to be tested for responsiveness to a
specific CDK7 inhibitor (e.g., Compound 1), but all samples are
measured for MYC, CDK18, CDK19, CCNE1, or FGFR1 enhancer strength.
In some embodiments, the samples are rank ordered based on MYC,
CDK18, CDK19, CCNE1, or FGFR1 enhancer strength. The choice of
which of the three methods set forth above to use to establish the
cutoff will depend upon the difference in MYC, CDK18, CDK19, CCNE1,
or FGFR1 enhancer strength between the lowest responder and the
highest non-responder in the population and whether the goal is to
minimize the number of false positives or to minimize the chance of
missing a potentially responsive sample or subject. When the
difference between the lowest responder and highest non-responder
is large (e.g., when there are many samples not tested for
responsiveness that fall between the lowest responder and the
highest non-responder in a rank ordering of MYC, CDK18, CDK19,
CCNE1, or FGFR1 enhancer strength), the cutoff is typically set
equal to or is up to 5% above the MYC, CDK18, CDK19, CCNE1, or
FGFR1 enhancer strength in the lowest responder in the population.
This cutoff maximizes the number of potential responders. When this
difference is small (e.g., when there are few or no samples
untested for responsiveness that fall between the lowest responder
and the highest non-responder in a rank ordering of MYC, CDK18,
CDK19, CCNE1, or FGFR1 enhancer strength), the cutoff is typically
set to a value in between the MYC, CDK18, CDK19, CCNE1, or FGFR1
enhancer strength of the lowest responder and the highest
non-responder. This cutoff minimizes the number of false positives.
When the highest non-responder has a MYC, CDK18, CDK19, CCNE1, or
FGFR1 enhancer strength that is greater than the lowest responder,
the cutoff is typically set to a value equal to or up to 5% above
the MYC, CDK18, CDK19, CCNE1, or FGFR1 enhancer strength in the
highest non-responder in the population. This method also minimizes
the number of false positives.
[0153] In some embodiments, the methods discussed above can be
employed to simply determine if a diseased cell (e.g., a cancer
cell) from a subject has a SE associated with a biomarker as
described herein (e.g., MYC, CDK18, CDK19, CCNE1, or FGFR1 or a
protein encoded thereby). The presence of the SE indicates that the
subject is likely to respond well to a covalent CDK7 inhibitor
(e.g., Compound 1). The cell is determined to have a SE associated
with the biomarker (e.g., MYC, CDK18, CDK19, CCNE1, or FGFR1 or a
protein encoded thereby) when the enhancer has a strength that is
equal to or above the enhancer associated with MALAT-1. In
alternate embodiments, the cell is determined to have a SE
associated with MYC, CDK18, CDK19, CCNE1, or FGFR1 when the MYC,
CDK18, CDK19, CCNE1, or FGFR1-associated enhancer has a strength
that is at least 10-fold greater than the median strength of all of
the enhancers in the cell. In other embodiments, the cell is
determined to have a SE associated with MYC, CDK18, CDK19, CCNE1,
or FGFR1 when the MYC, CDK18, CDK19, CCNE1, or FGFR1-associated
enhancer has a strength that is above the point where the slope of
the tangent is 1 in a rank-ordered graph of strength of each of the
enhancers in the cell.
[0154] In embodiments involving CDK18, the cutoff value for
enhancer strength can be converted to a prevalence cutoff, which
can then be applied to CDK18 mRNA levels to determine a mRNA cutoff
value in a given mRNA assay.
[0155] In some embodiments, mRNA levels of various genes of
interest according to this invention (e.g., BCL-XL, CDK7, CDK9,
CDK18, CDK19, CCNE1 or RB1) are used to determine sensitivity to a
covalent CDK7 inhibitor (e.g., Compound 1).
[0156] In some embodiments, gene of interest/biomarker mRNA levels
in a subject (as assessed, e.g., in a biological sample obtained
from the subject) are compared, using the same assay, to the same
gene of interest/biomarker mRNA levels in a population of subjects
having the same disease or condition to identify likely responders
to a covalent CDK7 inhibitor (a compound of Formula A or Compound
1). In embodiments where a biomarker is one whose mRNA expression
correlates with responsiveness to Compound 1 (e.g., CDK18, CDK19,
and CCNE1), at least some of the samples in the population will
have been tested for responsiveness to the inhibitor (e.g.,
Compound 1) to establish: (a) the lowest mRNA level of a sample in
the population that responds to that specific Compound 1 ("lowest
mRNA responder"); and, optionally, (b) the highest mRNA level of a
sample in the population that does not respond to that specific
Compound 1 ("highest mRNA non-responder"). In these embodiments, a
cutoff of biomarker mRNA level above which a test cell would be
considered responsive to that specific Compound 1 is set: i) equal
to or up to 5% above the mRNA level in the lowest mRNA responder in
the population; or ii) equal to or up to 5% above the mRNA level in
the highest mRNA non-responder in the population; or iii) a value
in between the mRNA level of the lowest mRNA responder and the
highest mRNA non-responder in the population.
[0157] In embodiments where mRNA levels positively correlate with
sensitivity to Compound 1, not all of the samples in a population
need to be tested for responsiveness to a Compound 1, but all
samples are measured for the gene of interest mRNA levels. In some
embodiments, the samples are rank ordered based on gene of interest
mRNA levels. The choice of which of the three methods set forth
above to use to establish the cutoff will depend upon the
difference in gene of interest mRNA levels between the lowest mRNA
responder and the highest mRNA non-responder in the population and
whether the cutoff is designed to minimize false positives or
maximize the potential number of responders. When this difference
is large (e.g., when there are many samples not tested for
responsiveness that fall between the lowest mRNA responder and the
highest mRNA non-responder in a rank ordering of mRNA levels), the
cutoff is typically set equal to or up to 5% above the mRNA level
in the lowest mRNA responder. When this difference is small (e.g.,
when there are few or no samples untested for responsiveness that
fall between the lowest mRNA responder and the highest mRNA
non-responder in a rank ordering of mRNA levels), the cutoff is
typically set to a value in between the mRNA levels of the lowest
mRNA responder and the highest mRNA non-responder. When the highest
mRNA non-responder has a mRNA level that is greater than the lowest
mRNA responder, the cutoff is typically set to a value equal to or
up to 5% above the mRNA levels in the highest mRNA non-responder in
the population.
[0158] In embodiments where a gene of interest/biomarker is one
whose mRNA expression inversely correlates with responsiveness to
Compound 1 (i.e., BCL-XL, CDK7, CDK9, and RB1), at least some of
the samples in the population will have been tested for
responsiveness to Compound 1 in order to establish: (a) the highest
mRNA level of a sample in the population that responds to that
specific Compound 1 ("highest mRNA responder"); and, optionally,
(b) the lowest mRNA level of a sample in the population that does
not respond to that specific Compound 1 ("lowest mRNA
non-responder"). In these embodiments, a cutoff of mRNA level above
which a test cell would be considered responsive to that specific
Compound 1 is set: i) equal to or up to 5% below the mRNA level in
the highest mRNA responder in the population; or ii) equal to or up
to 5% below the mRNA level in the lowest mRNA non-responder in the
population; or iii) a value in between the mRNA level of the lowest
mRNA non-responder and the highest mRNA responder and in the
population.
[0159] In embodiments where mRNA levels inversely correlate with
sensitivity to Compound 1, not all of the samples in a population
need to be tested for responsiveness to a Compound 1, but all
samples are measured for the gene of interest mRNA levels. In some
embodiments, the samples are rank ordered based on gene of interest
mRNA levels. The choice of which of the three methods set forth
above to use to establish the cutoff will depend upon the
difference in gene of interest mRNA levels between the highest mRNA
responder and the lowest mRNA non-responder in the population and
whether the cutoff is designed to minimize false positives or
maximize the potential number of responders. When this difference
is large (e.g., when there are many samples not tested for
responsiveness that fall between the highest mRNA responder and the
lowest mRNA non-responder in a rank ordering of mRNA levels), the
cutoff is typically set equal to or up to 5% below the mRNA level
in the highest mRNA responder. When this difference is small (e.g.,
when there are few or no samples untested for responsiveness that
fall between the highest mRNA responder and the lowest mRNA
non-responder in a rank ordering of mRNA levels), the cutoff is
typically set to a value in between the mRNA levels of the highest
mRNA responder and the lowest mRNA non-responder. When the highest
mRNA responder has a mRNA level that is lower than the lowest mRNA
responder, the cutoff is typically set to a value equal to or up to
5% below the mRNA levels in the lowest mRNA non-responder in the
population.
[0160] In embodiments involving CDK18, the cutoff for CDK18 mRNA
levels may be determined using the prevalence cutoff established
based on CDK18 enhancer strength, as described above. In some
aspects of these embodiments, a population is measured for mRNA
levels and the prior determined prevalence cutoff is applied to
that population to determine an mRNA cutoff level. In some aspects
of these embodiments a rank-order standard curve of CDK18 mRNA
levels in a population is created, and the pre-determined
prevalence cutoff is applied to that standard curve to determine
the CDK18 mRNA cutoff level.
[0161] In some aspects of embodiments where a test cell or sample
is compared to a population, the cutoff mRNA level value(s)
obtained for the population is converted to a prevalence rank and
the mRNA level cutoff is expressed as a percent of the population
having the cutoff value or higher, e.g., a prevalence cutoff.
[0162] Without being bound by theory, applicants believe that the
prevalence rank of a test sample and the prevalence cutoff in a
population will be similar regardless of the methodology used to
determine mRNA levels.
[0163] A subject can be identified as likely to respond well to a
covalent CDK7 inhibitor (e.g., a compound of Formula A or Compound
1) if the state of MYC, CDK18, CDK19, CCNE1, or FGFR1 (as
determined by, e.g., mRNA levels in a biological sample from the
subject) corresponds to (e.g., is equal to or greater than) a
prevalence rank in a population of about 80%, 79%, 78%, 77%, 76%,
75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,
62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%, 42%, 51%, 50%,
49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%,
36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%,
23%, 22%, 21%, or 20% as determined by the state of MYC, CDK18,
CDK19, CCNE1, or FGFR1, respectively, determined by assessing the
same parameter (e.g., mRNA level(s)) in the population. A subject
can be identified as likely to respond well to a covalent CDK7
inhibitor (e.g., a compound of Formula A or Compound 1) if the
state of BCL-XL, CDK7 or CDK9 (as determined by, e.g., mRNA levels
in a biological sample from the subject) is below a prevalence rank
in a population of about 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%,
72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%,
59%, 58%, 57%, 56%, 55%, 54%, 43%, 42%, 51%, 50%, 49%, 48%, 47%,
46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%,
33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or
20% as determined by the state of BCL-XL, CDK7 or CDK9,
respectively, determined by assessing the same parameter (e.g.,
mRNA level(s)) in the population. In some embodiments, the cutoff
value or threshold is established based on the biomarker (e.g.,
mRNA) prevalence value.
[0164] In still other embodiments, a population may be divided into
three groups: responders, partial responders and non-responders,
and two cutoff values (or thresholds) or prevalence cutoffs are set
or determined. The partial responder group may include responders
and non-responders as well as those subjects whose response to a
covalent CDK7 inhibitor (e.g., Compound 1) was not as high as the
responder group. This type of stratification may be particularly
useful when, in a population, the highest mRNA non-responder has an
mRNA level that is greater than that of the lowest mRNA responder.
In this scenario, for CDK18 or CDK19, the cutoff level or
prevalence cutoff between responders and partial responders is set
equal to or up to 5% above the CDK18 or CDK19 mRNA level of the
highest CDK18 or CDK19 mRNA non-responder; and the cutoff level or
prevalence cutoff between partial responders and non-responders is
set equal to or up to 5% below the CDK18 or CDK19 mRNA level of the
lowest CDK18 or CDK19 mRNA responder. For BCL-XL, CDK7 or CDK9,
this type of stratification may be useful when the highest mRNA
responder has a mRNA level that is lower than that of the lowest
mRNA non-responder. In this scenario, for BCL-XL, CDK7 or CDK9, the
cutoff level or prevalence cutoff between responders and partial
responders is set equal to or up to 5% below the mRNA level of the
lowest mRNA non-responder; and the cutoff level or prevalence
cutoff between partial responders and non-responders is set equal
to or up to 5% above the mRNA level of the highest mRNA responder.
The determination of whether partial responders should be
administered a covalent CDK7 inhibitor (e.g., Compound 1) will
depend upon the judgment of the treating physician and/or approval
by a regulatory agency.
[0165] Methods that can be used to quantify specific RNA sequences
in a biological sample are known in the art and include, but are
not limited to, fluorescent hybridization such as utilized in
services and products provided by NanoString Technologies, array
based technology (Affymetrix), reverse transcriptase qPCR as with
SYBR.RTM. Green (Life Technologies) or TaqMan.RTM. technology (Life
Technologies), RNA sequencing (e.g., RNA-seq), RNA hybridization
and signal amplification as utilized with RNAscope.RTM. (Advanced
Cell Diagnostics), or Northern blot. In some cases, mRNA expression
values for various genes in various cell types are publicly
available (see, e.g., broadinstitute.org/ccle; and Barretina et
al., Nature, 483:603-607, 2012).
[0166] In some embodiments, the state of a biomarker (as assessed,
for example, by the level of RNA transcripts) in both the test
biological sample and the reference standard or all members of a
population is normalized before comparison. Normalization involves
adjusting the determined level of an RNA transcript by comparison
to either another RNA transcript that is native to and present at
equivalent levels in both of the cells (e.g., GADPH mRNA, 18S RNA),
or to a fixed level of exogenous RNA that is "spiked" into samples
of each of the cells prior to super-enhancer strength determination
(Loven et al., Cell, 151(3):476-82, 2012; Kanno et al., BMC
Genomics 7:64, 2006; Van de Peppel et al., EMBO Rep., 4:387-93,
2003).
[0167] A subject (e.g., a human) suffering from a cancer described
herein may have been determined to be resistant (or to be acquiring
resistance after some initial efficacy) to a therapeutic agent that
was administered prior to the covalent CDK7 inhibitor (e.g.,
Compound 1). That prior therapeutic agent may be a platinum-based
anti-cancer agent administered as a monotherapy or in combination
with a standard of care.
[0168] Most cancer subjects eventually develop resistance to
platinum-based therapies by one or more of the following
mechanisms: (i) molecular alterations in cell membrane transport
proteins that decrease uptake of the platinum agent; (ii) molecular
alterations in apoptotic signaling pathways that prevent a cell
from inducing cell death; (iii) molecular alterations of certain
genes (e.g. BRCA1/2, CHEK1, CHEK2, RAD51) that restore the ability
of the cell to repair platinum agent-induced DNA damage. K. N.
Yamamoto et al., 2014, PloS ONE 9(8):e105724. The term "molecular
alterations" includes increased or decreased mRNA expression from
the genes involved in these functions; increased or decreased
expression of protein from such genes; and mutations in the
mRNA/proteins expressed from those genes.
[0169] Resistance is typically determined by disease progression
(e.g., an increase in tumor size and/or numbers) during treatment
or a decrease in the rate of shrinkage of a tumor. In some
instances, a patient will be considered to have become resistant to
a platinum-based agent when the patient's cancer responds or
stabilizes while on treatment, but which progresses within 1-6
months following treatment with the agent. Resistance can occur
after any number of treatments with platinum agents. In some
instances, disease progression occurs during, or within 1 month of
completing treatment. In this case, the patient is considered to
have never demonstrated a response to the agent. This is also
referred to a being "refractory" to the treatment. Resistance may
also be determined by a treating physician when the platinum agent
is no longer considered to be an effective treatment for the
cancer.
[0170] In some embodiments, the subject is, has been determined to
be, or has become resistant to treatment with a CDK4/6 inhibitor
administered as a monotherapy or in combination with a standard of
care.
[0171] CDK4/6 inhibitors in cancer (e.g., HR.sup.+ breast cancer)
are known to block entry into S phase of the cell cycle by inducing
G1 arrest. Resistance to CDK4/6 inhibitors in cancer (e.g.,
HR.sup.+ metastatic breast cancer) has been shown to be mediated,
in part, by molecular alterations that: 1) enhance CDK4/6 activity,
such as amplifications of CDK6, CCND1, or FGFR1 (Formisano et al.,
SABCS 2017, Publication Number GS6-05; Cruz et al., SABCS 2017
Publication Number PD4-05), or 2) reactivate cell cycle entry
downstream of CDK4/6, such as RB1 loss and CCNE1 amplification
(Condorelli, Ann Oncol, 2017 PMID: 29236940; Herrera-Abreu MT,
Cancer Research 2016 PMID: 27020857). As shown in FIG. 35,
responses to Compound 1 in ovarian PDX models was associated with
low RB1 expression or high CCNE1 expression. Further genetic and
molecular analyses of these ovarian PDXs has revealed additional
features in support of the antitumor activity of Compound 1 in
tumors with molecular alterations that otherwise confer CDK4/6
inhibitor resistance (see Example 6).
[0172] In addition, Compound 1 inhibits many HR.sup.+ breast cancer
cells in vitro, including those with a form of acquired resistance
to aromatase inhibitors, as well as those that no longer respond to
inhibitors of CDK4/6 (data not shown). Fulvestrant is a second line
standard of care for breast cancer patients who have failed
aromatase inhibitor treatment. In addition, it is known that the
CDK7 inhibitor THZ-1 showed in vitro synergy with fulvestrant in
several HR.sup.+ breast cancer cell lines (Jeselsohn et al., Cancer
Cell, 33:173-86, 2018). This provides a mechanistic rationale for
the efficacy of Compound 1 in combination with fulvestrant in
patients with HR.sup.+ metastatic breast cancer who have progressed
following treatment with a CDK4/6 inhibitor plus an aromatase
inhibitor.
[0173] Unlike platinum-based agents which are typically
administered for a period of time followed by a period without
treatment, CDK4/6 inhibitors, such as palbociclib, ribociclib or
abemaciclib, are administered until disease progression is
observed. In some instances, a patient will be considered to have
become resistant to a CDK4/6 inhibitor when the patient's cancer
initially responds or stabilizes while on treatment, but which
ultimately begins to progress while still on treatment. In some
instances, a patient will be considered to be resistant (or
refractory) to treatment with a CDK4/6 inhibitor if the cancer
progresses during treatment without demonstrating any significant
response or stabilization. Resistance may also be determined by a
treating physician when the CDK4/6 inhibitor is no longer
considered to be an effective treatment for the cancer.
[0174] A covalent CDK7 inhibitor (e.g., a compound of Formula A,
Compound 1 or a pharmaceutically acceptable salt thereof) and any
second therapeutic agent utilized in the methods described herein
can be included in a kit and/or formulated in a pharmaceutically
acceptable composition that includes the first agent, the second
agent, and a pharmaceutically acceptable carrier.
[0175] A covalent CDK7 inhibitor (e.g., Compound 1) and any second
therapeutic agent utilized in the methods of the present disclosure
can be prepared and administered in a wide variety of oral or
parenteral dosage forms. Thus, these agents can be administered by
injection (e.g. intravenously, intramuscularly, intracutaneously,
subcutaneously, intraduodenally, or intraperitoneally).
Alternatively, Compound 1 and any second therapeutic agent can be
administered by inhalation, for example, intranasally.
Additionally, Compound 1 and any second therapeutic agent can be
administered transdermally. It is also envisioned that multiple
routes of administration (e.g., intramuscular, oral, transdermal)
can be used to administer one or both of Compound 1 and the second
therapeutic agent.
[0176] For preparing pharmaceutical compositions including a
compound described herein, pharmaceutically acceptable excipients
can be added in either solid or liquid form or a combination
thereof. Solid form preparations within the scope of the present
invention include powders, tablets, pills, capsules, cachets,
suppositories, and dispersible granules. A solid carrier can be a
substance that may also act as a diluent, flavoring agent, binder,
preservative, tablet disintegrating agent, or encapsulating
material. In powders, the excipient (e.g., a carrier) is a finely
divided solid in a mixture with the finely divided active component
(e.g., a compound described herein). In tablets, the active
component (e.g., a compound described herein) is mixed with the
excipient having the necessary binding properties in suitable
proportions and compacted in the shape and size desired.
Pharmaceutical compositions, including those formulated as powders
and tablets, can contain from 5% to 70% of the active compound
(i.e., a compound described herein). Suitable excipients (e.g.,
carriers) are magnesium carbonate, magnesium stearate, talc, sugar,
lactose, pectin, dextrin, starch, gelatin, tragacanth,
methylcellulose, sodium carboxymethylcellulose, a low melting wax,
cocoa butter, and the like. The term "preparation," when used in
connection with a pharmaceutical composition, is intended to
include, but is not limited to, the formulation of an active
compound with encapsulating material as a carrier providing a
capsule in which the active component with or without other
carriers, is surrounded by a carrier, which is thus in association
with it. Similarly, cachets and lozenges are included. Tablets,
powders, capsules, pills, cachets, and lozenges can be used as
solid dosage forms suitable for oral administration.
[0177] For preparing suppositories, a low melting wax, such as a
mixture of fatty acid glycerides or cocoa butter, is first melted
and the active component is dispersed homogeneously therein, as by
stirring. The molten homogeneous mixture is then poured into
convenient sized molds, allowed to cool, and thereby to
solidify.
[0178] Liquid form preparations include solutions, suspensions, and
emulsions, for example, water or water/propylene glycol solutions.
For parenteral injection, liquid preparations can be formulated in
solution in aqueous polyethylene glycol solution.
[0179] When parenteral application is needed or desired,
particularly suitable admixtures for the compounds of the invention
are injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. In some embodiments, suitable carriers for
parenteral administration will be selected for human
administration. In particular, carriers for parenteral
administration include aqueous solutions of dextrose, saline, pure
water, ethanol, glycerol, glycerol formal, polyethylene glycol,
propylene glycol, peanut oil, sesame oil, polyoxyethylene-block
polymers, pyrrolidine, N-methyl pyrrolidione, and the like.
Ampoules are convenient unit dosages. The compounds of the present
disclosure can also be incorporated into liposomes or administered
via transdermal pumps or patches. Pharmaceutical admixtures
suitable for use in the present disclosure include those described,
for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co.,
Easton, Pa.) and WO 96/05309, the teachings of both of which are
hereby incorporated by reference.
[0180] Aqueous solutions suitable for oral use can be prepared by
dissolving the active component in water and adding suitable
colorants, flavors, stabilizers, and thickening agents as desired.
Aqueous suspensions suitable for oral use can be made by dispersing
the finely divided active component in water with viscous material,
such as natural or synthetic gums, resins, methylcellulose, sodium
carboxymethylcellulose, and other well-known suspending agents.
[0181] Also included are solid form preparations that are intended
to be converted, shortly before use, to liquid form preparations
for oral administration. Such liquid forms include solutions,
suspensions, and emulsions. These preparations may contain, in
addition to the active component, colorants, flavors, stabilizers,
buffers, artificial and natural sweeteners, dispersants,
thickeners, solubilizing agents, and the like.
[0182] The pharmaceutical compositions can be in unit dosage forms.
In such form, the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged form.
[0183] The quantity of active component in a unit dose preparation
may be similar to that being utilized in a human clinical trial or,
for those agents that have already been approved for use, the
dosages indicated on the prescribing information for that agent (or
dosages below those described in the prescribing information where
a synergistic effect is attained).
[0184] Some compounds may have limited solubility in water and
therefore may require a surfactant or other appropriate co-solvent
in the composition. Such co-solvents include: Polysorbate 20, 60,
and 80; Pluronic F-68, F-84, and P-103; cyclodextrin; and polyoxyl
35 castor oil. Such co-solvents are typically employed at a level
between about 0.01% and about 2% by weight.
[0185] Viscosity greater than that of simple aqueous solutions may
be desirable to decrease variability in dispensing the
formulations, to decrease physical separation of components of a
suspension or emulsion of formulation, and/or otherwise to improve
the formulation. Such viscosity building agents include, for
example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl
cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose,
carboxymethyl cellulose, hydroxy propyl cellulose, chondroitin
sulfate and salts thereof, hyaluronic acid and salts thereof, and
combinations of the foregoing. Such agents are typically employed
at a level between about 0.01% and about 2% by weight.
[0186] Pharmaceutical compositions of the present invention may
additionally include components to provide sustained release and/or
comfort (e.g., high molecular weight, anionic mucomimetic polymers,
gelling polysaccharides, and finely-divided drug carrier
substrates). These components are discussed in greater detail in
U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The
entire contents of these patents are incorporated herein by
reference in their entirety for all purposes.
[0187] For many of the agents described herein, effective dosage
forms are known in the art.
[0188] A covalent CDK7 inhibitor described herein, (e.g., Compound
1) can be formulated into an aqueous pharmaceutical composition
comprising sulfobutyl ether-.beta.-cyclodextrin (SBE.beta.CD), such
as Captisol.RTM. and/or formulated into a dosage form for
intravenous infusion.
[0189] Pharmaceutical compositions utilized in the present
disclosure include compositions wherein the active ingredient(s) is
contained in a therapeutically effective amount, i.e., in an amount
effective to achieve its intended purpose. The actual amount
effective for a particular application will depend, inter alia, on
the condition being treated. For example, when administered in
methods to a subject with cancer, such compositions will contain an
amount of active ingredient effective to achieve the desired
result.
[0190] The dosage and frequency (single or multiple doses) of
compound administered can vary depending upon a variety of factors,
including route of administration; size, age, sex, health, body
weight, body mass index, and diet of the recipient; nature and
extent of symptoms of the disease being treated; presence of other
diseases or other health-related problems; kind of concurrent
treatment; and complications from any disease or treatment regimen.
Other therapeutic regimens or agents can be used in conjunction
with the methods and compounds of the present disclosure.
[0191] For any compound or pharmaceutical composition described
herein, the therapeutically effective amount can be initially
determined from, or informed by data generated in, cell culture
assays and/or animal models of disease. For example, a dose for
humans can be formulated to achieve a concentration that has been
found to be effective in animals. The dosage in humans can be
adjusted by, for example, monitoring kinase inhibition, other
markers, the signs and symptoms of the disease being treated, and
side effects and subsequently adjusting the dosage upwards or
downwards.
[0192] Dosages may be varied depending upon the requirements of the
patient and the compound being employed. The dose administered to a
patient, in the context of the present disclosure, should be
sufficient to effect a beneficial therapeutic response in the
patient over time. The size of the dose also will be determined by
the existence, nature, and extent of any adverse side effects.
Generally, treatment is initiated with smaller dosages, which are
less than the optimum dose of the compound. Thereafter, the dosage
is increased by small increments until the optimum effect under
circumstances is reached.
[0193] For many of the agents described herein, effective dosage
amounts and intervals are known in the art. Such dosage amounts and
intervals can be adjusted individually to provide levels of the
administered compound(s) effective for the particular clinical
indication being treated. This will provide a therapeutic regimen
that is commensurate with the severity of the individual's disease
state.
[0194] In one embodiment, the amount of the covalent CDK7 inhibitor
(e.g., Compound 1 or a compound of Formula A) to be administered is
between about 1-500 mg/m.sup.2 administered once or twice per week.
For example, the amount to be administered can be between 2-128
mg/m.sup.2 once or twice a week via intravenous infusion.
[0195] In certain embodiments, the invention provides the use of a
combination of Compound 1 and a second therapeutic agent selected
from a) a Bcl-2 inhibitor, b) a CDK9 inhibitor, c) a Flt3
inhibitor, d) a PARP inhibitor, e) a BET inhibitor, f) a CDK4/6
inhibitor, g) a platinum-based anti-cancer agent, or h) a taxane to
treat a subject suffering from a disease (e.g., a cancer; e.g., a
specific type of cancer, e.g., a breast cancer or an ovarian
cancer). Inhibitors in each of these classes of second therapeutic
agents are well-known in the art. In some aspects of these
embodiments, the subject to be treated is naive (e.g., has not been
exposed to) the second therapeutic agents. In alternate aspects of
these embodiments, the subject to be treated has been exposed to
and has demonstrated resistance or is refractory to the second
therapeutic agent when administered as a monotherapy.
[0196] Examples of useful Bcl-2 inhibitors include, but are not
limited to, venetoclax, APG-1252, S55746, BP1002, and APG-2575.
[0197] Examples of useful CDK9 inhibitors include, but are not
limited to, alvocidib, seliciclib (CYC202), AT7519, TG02, CYC065,
BAY1251152, BAY 1143572, voruciclib (formerly P1446A-05), TP-1287,
AZD5576, NVP2, nanoflavopiridol, and VS2-370.
[0198] Examples of useful Flt3 inhibitors include, but are not
limited to, Rydapt.RTM. (midostaurin), Quizartinib,
Pexidartinib/PLX3397, gilteritinib (ASP2215), Crenolanib besylate,
Nexavar.RTM. (sorafenib), CDX-301, Iclusig.RTM. (ponatinib),
pacritinib, SEL24, ENMD-2076, FF-10101-01, CT053PTSA, SKI-G-801,
SKLB1028, FLYSYN, NMS-088, CG'806, and HM43239.
[0199] Examples of useful PARP inhibitors include, but are not
limited to, Lynpraza.RTM. (olaparib), Zejula.RTM. (niraparib),
Rubraca.RTM. (rucaparib), veliparib, talazoparib, 2x-121, CK-102,
BGB-290, NT-125, and NMS-P293.
[0200] Examples of useful BET inhibitors include, but are not
limited to, JQ1, GS-5829, FT-1101, ZEN-3694, GSK-2820151, I-BET762,
GSK525762, CPI-0610, OTX015, I-BET151, CPI203, PFI-1, MS436,
RVX2135, BAY1238097, INCB054329, TEN-010, BAY-299, BMS-986158,
ABBV-075, and PLX51107.
[0201] Examples of useful CDK4/6 inhibitors include, but are not
limited to, Ibrance.RTM. (palbociclib), Kisqali.RTM. (ribociclib),
Verzenio.RTM. (abemaciclib), trilaciclib, G1T38, BPI-1178, and ON
123300.
[0202] Examples of useful platinum-based anti-cancer agents include
cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin
tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
[0203] Examples of useful taxanes include Cremophor EL-paclitaxel
(Taxol.RTM.), nab-paclitaxel (Abraxane.RTM.), and docetaxel
(Taxotere.RTM.).
[0204] Unless otherwise specified, when employing a combination of
a covalent CDK7 inhibitor (e.g., Compound 1 or a compound of
Formula (A)) and a second therapeutic agent in a method of the
invention, the second therapeutic agent can be administered
concurrently with, prior to, or subsequent to the covalent CDK7
inhibitor. The second therapeutic pharmaceutical agent may be
administered at a dose and/or on a time schedule determined for
that pharmaceutical agent. The second therapeutic agent may also be
administered together with the covalent CDK7 inhibitor (e.g.,
Compound 1) in a single dosage form or administered separately in
different dosage forms. In general, it is expected that the second
therapeutic agents utilized in combination with Compound 1 will be
utilized at levels that do not exceed the levels at which they are
utilized individually. In some embodiments, the levels of the
second therapeutic agent utilized in combination will be lower than
those utilized in a monotherapy due to synergistic effects.
[0205] For combinations of a covalent CDK7 inhibitor (e.g.,
Compound 1 or a compound of Formula A) and a second therapeutic
agent, a kit comprising each of the two active therapeutics can be
provided. In some instances, each of Compound 1 and the second
therapeutic agent will be in separate vessels. In some instances,
the kit includes a written insert or label with instructions to use
the two therapeutics in a subject suffering from a cancer (e.g., as
described herein). The instructions may be adhered or otherwise
attached to a vessel or vessels comprising the therapeutic agents.
Alternatively, the instructions and the vessel(s) can be separate
from one another but present together in a single kit, package,
box, or other type of container.
[0206] The instructions in the kit will typically be mandated or
recommended by a governmental agency approving the therapeutic use
of the combination. The instructions may optionally comprise dosing
information for each therapeutic agent, the types of cancer for
which treatment of the combination was approved or is intended,
physicochemical information about each of the therapeutics,
pharmacokinetic information about each of the therapeutics,
drug-drug interaction information, or diagnostic information (e.g.,
based on a biomarker described herein).
EXAMPLES
Example 1. Correlating Compound 1 Sensitivity, Gene Expression
and/or SE Strength
[0207] To identify potential biomarkers predictive of sensitivity
to Compound 1, we evaluated the inhibitory activity of Compound 1
in a large panel of human tumor cell lines and correlated activity
with RNA expression and epigenetic profiling data.
[0208] A panel of 406 human tumor cell lines (Chempartner),
including 19 TNBC cell lines, were tested for response to various
concentrations of Compound 1 (0.0005, 0.0015, 0.0046, 0.014, 0.041,
0.12, 0.37, 1.1, 3.3, and 10 uM) using the ATP-lite assay. Cell
line growth was measured before treatment on Day 0 and then after
treatment either after a minimum of 3 or maximum of 6 days
depending on the cell-line. Simultaneously, the signal was for the
same cell-line treated with DMSO for the same number of days as a
negative control.
[0209] Clustering of growth-rate adjusted dose response curves of
cell-lines treated with Compound 1 allowed the classification of
cell-lines into low and high response groups. Based on the assay
values, we computed normalized growth rate inhibition values at
each concentration of Compound 1 by comparing growth rates in the
presence and absence of that drug and then fit the results to a
sigmoidal curve using known methods (Hafner et al., Nature Methods,
2016).
[0210] Fitting of the growth rate inhibition curves (GR curves)
enabled measurement of various metrics such as GRmax (the maximum
GR at the highest concentration of drug tested), and GRO (the
concentration of drug at which the GR value crossed 0 (transitioned
from cytostatic to cytotoxic)).
[0211] We next assigned each cell line to either the "high" or
"low" categories based on response to Compound 1. We developed the
following algorithm for this purpose. We assigned two sets of
seeds: a low responder group of cell-lines all with GRmax>0
(e.g., cell lines for which Compound 1 was not cytotoxic even at
the highest concentration tested), and a high responder group with
GRmax<-0.5 and GRO<100 nM (e.g., cell lines for which
Compound 1 was highly cytotoxic at a concentration of <100 nM).
The goal of this algorithm was to classify all the remaining
cell-lines that did not fall into one of these two groups as "low"
or "high" responders, while allowing for some flexibility in
reassigning the seeds to a different group. Using pairwise
Euclidean distance between the theoretical GR curves for all
cell-lines, we performed hierarchical clustering with Ward.D2
linkage 1000 times (clusterings), using a random subset of 90% of
the cell lines each time. For each clustering, a cell line was
counted as "high" if its curve was closer to high response seed and
"low" if its curve was closer to low response seed in that
particular clustering iteration. We then determined the % of
clustering iterations that the cell line was counted as "high" and
"low". If the cell line was counted as "low" in over 50% of the
iterations, it was classified as a "low" responder. If the cell
line was counted as "high" in over 50% of the iterations, it was
classified as being a "high" responder.
[0212] For all cell lines assigned to low and high responder
categories, we identified those that had matching mRNA expression
data, either microarray or RNA-Seq, from Cancer Cell Line
Encyclopedia (CCLE). We identified 18,074 genes with both
microarray and RNA-seq data available. For each gene and each cell
line mRNA dataset, we built a linear classifier to predict whether
the cell line belonged to the low responder class or high responder
class based on the mRNA expression level of only that gene. The
classifier used was the linear discriminant analysis from the MASS
package in R and the assignment of each cell line was tested using
leave-one-out cross validation. True positive rate was calculated
as the number of high responder cell-lines that were accurately
classified as such based solely on mRNA expression, while true
negative rate was calculated as the number of low responder cell
lines that were accurately classified as such based solely on mRNA
expression. "Accuracy" was measured as the average of true positive
rate and true negative rate. False Discovery Rate (FDR) was
calculated by randomly permuting "low" and "high" responder labels
across cell-lines 1000 times, and then calculating the accuracy for
each iteration using the linear classifier-based approach described
above. We then approximated the distribution of random balanced
accuracies per gene as a normal distribution. FDR was calculated as
the probability of observing an accuracy value from the random
distribution that was higher than the real predicted accuracy for
that gene. The lower the FDR, the higher our confidence would be
that the gene expression was truly predictive of cell-line
response. Typically, an accuracy of at least 65% combined with an
FDR<0.05 indicated that expression of a particular gene was
predictive of sensitivity to Compound 1. Higher FDRs required
higher accuracy to find a correlation between mRNA expression (or
super-enhancer strength) sensitivity to Compound 1.
[0213] SE analysis was performed using genome-wide H3K27Ac scores
based on the SE score algorithm of McKeown et al. (Cancer
Discovery, 7(10):1136-1153, 2017). H3K27ac scores per SE were used
to calculate accuracy and FDR in the same way as described above
for expression per gene. Copy number data was obtained from
CCLE.
[0214] For the MYC CNV plot (FIG. 1B), we built a linear classifier
using the CNV values for the MYC gene to predict the "low" or
"high" responder class for cell-lines as described above.
[0215] Twenty-five genes were differentially expressed between
Compound 1-sensitive and -insensitive tumor lines (FDR<0.05
and/or accuracy>65%). The cell lines were further divided by
cancer type and/or subtype to determine if the differential
expression of these genes was significant in determining
sensitivity to Compound 1. Of the 25 genes, mRNA expression levels
of at least some of them predicted the response to Compound 1 in at
least a subset of cancers.
[0216] For MYC, there was no significant correlation between mRNA
expression and response to Compound 1 in the majority of cancer
subtypes (FIG. 1A), nor was there any significant correlation
between MYC copy number and sensitivity (FIG. 1B). A significant
correlation was found, however, between MYC-associated super
enhancer strength (as determined by SE score) and sensitivity to
Compound 1 in breast cancer samples tested and was also seen in
both TNBC and non-TNBC sample subsets (FIG. 1C). Moreover, further
analysis of TNBC cells demonstrated that there was a correlation
between MYC-associated super enhancer strength and MYC expression
as shown in FIG. 1D.
[0217] For CDK7, we found a significant inverse correlation between
CDK7 mRNA levels and sensitivity to Compound 1 in all samples and
in lymphoma. We also observed an inverse correlation between CDK7
mRNA levels and sensitivity to Compound 1 in lung cancer, leukemia,
and stomach cancer cell lines (FIG. 2).
[0218] For CDK9, we found a significant inverse correlation between
mRNA levels and sensitivity to Compound 1 in all breast and TNBC
cell lines (FIG. 3).
[0219] For CDK19, we found a significant correlation between mRNA
levels and sensitivity to Compound 1 in all, all breast cancer,
TNBC, all lung, small cell lung and non-small cell lung cancer cell
lines (FIG. 4).
[0220] For CDK18, we found a significant correlation between super
enhancer strength (FIG. 5A) and sensitivity to Compound 1 in all
breast and TNBC cell lines. We also found a significant correlation
between mRNA levels and sensitivity to Compound 1 in all breast and
TNBC cell lines (FIG. 5B).
[0221] For BCL2L1, we found a significant inverse correlation
between mRNA levels and sensitivity to Compound 1 in all cancer,
all breast cancer (Accuracy=73%; FDR=0.055), TNBC, non-TNBC,
ER.sup.+/PR.sup.+ breast cancer, HER2.sup.+ breast cancer, all lung
cancer, NSCLC, ovarian, leukemia and stomach cancer cell lines
(FIGS. 22A and 22B). Thus, lower expression of BCL2L1, which
encodes the mitochondrial apoptosis regulator BCL-XL, was
identified as the most predictive expression biomarker of
sensitivity across all profiled cell lines, strongly separating the
two classes of sensitivity.
[0222] The table below shows both the Accuracy and FDR values for
each biomarker and each type of cancer cell line tested against
Compound 1.
TABLE-US-00002 TABLE 1 Accuracy and FDR Values for Various
Biomarker Gene Expression in Various Cancer Cell Line Types Treated
with Compound 1. Indication Statistic MYC CDK7 CDK9 CDK18 CDK19
BCL2L1 All Accuracy (%) 49 62 52 55 61 70 FDR 0.52 0.051 0.39 0.24
0.075 0.0061 Lymphoma Accuracy (%) 58 82 63 37 63 48 FDR 0.33 0.028
0.2 0.77 0.21 0.55 Breast (all) Accuracy (%) 65 42 71 70 70 76 FDR
0.15 0.69 0.076 0.093 0.084 0.043 TNBC Accuracy (%) 66 57 73 80 82
54 FDR 0.21 0.33 0.1 0.051 0.058 0.41 Breast, Accuracy (%) 48 67 70
49 18 79 Non-TNBC FDR 0.54 0.18 0.14 0.53 0.95 0.076 Lung (All)
Accuracy (%) 63 69 53 59 72 65 FDR 0.15 0.064 0.4 0.21 0.029 0.12
Small Cell Accuracy (%) 0 43 43 64 65 43 Lung FDR 0.99 0.59 0.62
0.21 0.19 0.63 Non-Small Accuracy (%) 42 55 57 12 71 64 Cell Lung
FDR 0.7 0.36 0.32 0.99 0.099 0.16 Ovary Accuracy (%) 54 61 60 66 33
73 FDR 0.41 0.24 0.26 0.18 0.83 0.1 Pancreas Accuracy (%) 48 47 42
71 56 53 FDR 0.55 0.55 0.7 0.12 0.38 0.43 Leukemia Accuracy (%) 68
63 54 51 58 89 FDR 0.099 0.22 0.39 0.45 0.31 0.015 AML Accuracy (%)
81 28 41 3 25 94 FDR 0.05 0.86 0.66 0.98 0.87 0.014 Stomach
Accuracy (%) 50 72 67 61 50 72 FDR 0.49 0.11 0.19 0.23 0.49
0.13
[0223] To confirm that sensitivity to Compound 1 was not simply due
to some general toxic effect on the cells, we tested the
sensitivity of these cell lines to the general kinase inhibitor
staurosporine and queried if there was a correlation between
sensitivity and various of the above-described biomarker mRNA
level. We found no correlation between staurosporine sensitivity
and BCL-XL expression (FIG. 6). We found some inverse correlation
between staurosporine sensitivity and CDK7 expression, but not as
strong as between Compound 1 sensitivity and CDK7 expression (FIG.
7). For CDK9, we found a strong correlation between mRNA level and
staurosporine sensitivity in TNBC, just as we found for Compound 1
(FIG. 8). These results demonstrate that the correlations and
inverse correlations found between sensitivity to Compound 1 and
expression of various biomarkers is specific to the mechanism of
action of Compound 1 and not due to a toxic effect of that compound
on the cell lines.
[0224] The table below shows both the Accuracy and FDR values for
each biomarker and each type of cancer cell line tested against
staurosporine.
TABLE-US-00003 TABLE 2 Accuracy and FDR Values for Various
Biomarker Gene Expression in Various Cancer Cell Line Types.
Indication Statistic BCL2L1 CDK7 CDK9 All Accuracy (%) 54 55 50 FDR
0.34 0.16 0.52 Lymphoma Accuracy (%) 49 63 56 FDR 0.49 0.16 0.35
Breast (all) Accuracy (%) 57 57 85 FDR 0.28 0.29 0.0083
Triple-negative Accuracy (%) 67 51 92 Breast FDR 0.2 0.42 0.0068
Breast, Not TNBC Accuracy (%) 48 32 45 FDR 0.016 0.5 0.08 Lung
(All) Accuracy (%) 53 57 56 FDR 0.36 0.28 0.33 Small Cell Lung
Accuracy (%) 64 38 60 FDR 0.22 0.75 0.26 Non-Small Cell Accuracy
(%) 61 61 58 Lung FDR 0.2 0.17 0.3 Pancreas Accuracy (%) 37 51 35
FDR 0.77 0.39 0.76 Leukemia Accuracy (%) 66 42 50 FDR 0.18 0.68 0.5
AML Accuracy (%) 70 60 55 FDR 0.16 0.28 0.43 Bone Accuracy (%) 71
65 44 FDR 0.15 0.23 0.6 Stomach Accuracy (%) 65 14 57 FDR 0.22 0.97
0.34
[0225] The table below shows both the Accuracy and FDR values for
MYC and CDK18 SEs in breast cancer cell lines tested against
Compound 1.
TABLE-US-00004 TABLE 3 Accuracy and FDR Values for Various
Biomarker Gene Expression in Various Cancer Cell Line Types.
Accuracy FDR Accuracy FDR TNBC TNBC All Breast All Breast MYC SE
86% 0.017 76% 0.023 CDK18 SE 79% 0.065 70% 0.094
[0226] The table below shows both the Accuracy and FDR values for
MYC copy number in cancer cell lines tested against Compound 1.
TABLE-US-00005 TABLE 4 Accuracy and FDR Values for the Presence of
a MYC Super-Enhancer in Various Cancer Cell Line Types. Indication
Statistic MYC Lymphoma Accuracy (%) 72 FDR 0.08 Breast (all)
Accuracy (%) 60 FDR 0.23 TNBC Accuracy (%) 64 FDR 0.22 Breast, Not
TNBC Accuracy (%) 46 FDR 0.57 Lung (All) Accuracy (%) 45 FDR 0.63
Small Cell Lung Accuracy (%) 54 FDR 0.41 Non-Small Cell Accuracy
(%) 5 Lung FDR 1 Ovary Accuracy (%) 61 FDR 0.27 Pancreas Accuracy
(%) 45 FDR 0.63 Leukemia Accuracy (%) 26 FDR 0.94 Bone Accuracy (%)
78 FDR 0.09 Stomach Accuracy (%) 28 FDR 0.9
[0227] In these studies, we show for the first time that Compound 1
induced differential responses across a large panel of human tumor
cell lines derived from multiple indications. We also show that, in
this panel of cell lines, the response could be predicted in an
"indication agnostic" manner by the level of expression of BCL2L1.
Finally, in line with prior reports, in TNBC cell lines, MYC SE was
significantly associated with sensitivity to Compound 1. These
observations have generated strong hypotheses for selection
strategies aimed at identifying patients with tumors particularly
sensitive to CDK7 inhibition with Compound 1 and warrant further
investigation with respect to predictive biomarkers of response in
patients. Compound 1 is currently being assessed in a phase 1 trial
in adult patients with advanced solid tumors, including a planned
expansion cohort enriching for patients with TNBC
(NCT03134638).
Example 2. Compound 1 is Associated with Downregulation of MCL1 in
Breast Cancer and Ovarian Cancer Cell Lines
[0228] The inverse correlation between efficacy of Compound 1 and
BCL-XL mRNA level led us to hypothesize that Compound 1 was
affecting the intrinsic apoptotic pathway of which BCL-XL is a
part. It is known that three BCL2 family members, MCL1, BCL-XL and
BCL2 are all involved in inhibiting apoptosis with somewhat
redundant functions. Thus, we explored the effect of Compound 1 on
each of these genes at both the mRNA and protein level in various
cancer cell lines.
[0229] Cytotoxic cancer cell lines HCC70 and TOV21G, and cytostatic
cancer cell lines T47D and COV318 were seeded in wells of a
six-well dish at a density of 1.times.10.sup.6 cells/well and
allowed to adhere overnight. Cells were then treated with 50 nM of
Compound 1 for 16 or 24 hours, or with DMSO (0 hours) representing
a negative control. Cells were harvested and placed on ice, and
resuspended in RIPA lysis buffer supplemented with protease and
phosphatase inhibitors. Clarified lysates prepared with 4.times.LSD
running buffer and boiled at 95.degree. C. for 5 minutes.
Equivalent amounts of sample (10 .mu.g total protein) were run on a
4-12% Bis-Tris gel and transferred to a PDVF membrane for Western
blotting using standard Western blotting protocols. Membranes were
blocked with Licor TBS blocking buffer then probed with antibodies
for BCL-XL, BCL-2, MCL1, BID, and GAPDH visualized using and
Odyssey imager (FIGS. 23A and 23D).
[0230] Breast cancer cell lines HCC70, HCC38, T47D, and MDAMB231
were plated in a 96-well plate at 20,000 cells/well and allowed to
adhere overnight. Cells were then treated with 50 nM of Compound 1
or DMSO for 24 hours. Media was aspirated and mRNA isolated using
Dynabeads.TM. mRNA DIRECT.TM. Purification Kit (Invitrogen) and
amount of mRNA measured using qPCR with the TaqMan probes for the
transcript of interest (either BCLXL or MCL1). Transcripts were
quantified using AACt calculation, normalizing to the house keeping
gene GAPDH (FIGS. 23B and 23C).
[0231] In a similar experiment, TNBC breast cancer cell lines
HCC70, MDAMB468, MDAMB453 and CAL-120 were grown as described above
and treated with DMSO, 50 nM Compound 1 or 100 nM Compound 1 for 24
hours. Protein samples were prepared as described above for Western
blotting using antibodies specific for the indicated proteins in
FIG. 29A. As shown in FIG. 29A, MCL1 was downregulated by Compound
1 treatment in all four cell lines, but more substantially in cell
lines having low levels of BCLXL (HCC70 and MDAMB468) as compared
to the cell line having substantially higher levels of BCLXL
(CAL-120).
Example 3. Compound 1 is Associated with Downregulation of MCL1 in
AML Cell Lines Having Low BCL-XL and Higher BCL2
[0232] We next explored the expression of MCL1, BCL-XL and BCL2 in
four different AML cell lines. MV411, OCI-AML3 and KG1 cells were
separately grown and prepared as described in Example 2 for Western
blotting, using antibodies specific for BCL2 (FIG. 24A), BCL-XL,
MCL-1 and tubulin as an internal control (FIG. 24B).
[0233] As shown in FIG. 24A, BCL2 protein was robustly expressed in
three of the tested AML cell lines (MV411, OCI-AML3 and KG1), but
not in the fourth (OC1M1). Treatment of these four cell lines with
50 nM of Compound 1 resulted in significant reduction in MCL1 in
MV411, OCI-AML3 and KG1, but little effect on that target in OC1M1
(FIG. 24B). Not surprisingly, OCIM1 expressed a high amount of
BCL-XL compared to BCL-XL levels in the other three cell lines
(FIG. 24B). These results seem to confirm our findings that high
BCL-XL expression levels are correlated with low sensitivity to
Compound 1 and suggest that such high expression levels may affect
the ability of Compound 1 to decrease MCL1.
Example 4. Exploration of Synergy Between Compound 1 and Other
Cancer Therapeutics
[0234] The redundancy of the BCL2 family members' role as apoptosis
inhibitors and our results showing the inverse correlation between
the efficacy of Compound 1 and BCL-XL mRNA level suggested that
effective treatment of certain cancers might require low levels of
all three of BCL-XL, BCL2 and MCL1. We therefore examined the
combined effect of Compound 1 and the known BCL2 inhibitor
venetoclax on various cancer cell lines, as well as the combined
effect of Compound 1 and other therapeutic agents.
[0235] Using a Biotek EL406, 50 .mu.L of cell media containing
20-60,000 cells/ml was distributed into white 384-well Nunc plates
(Thermo). Suspension cells then received compound immediately while
adherent cells lines were given one hour to reattach to the surface
of the plate prior to compound addition. Compound 1 and the second
agents to be tested were dissolved in DMSO and arrayed on 384 well
compound storage plates (Greiner). Each compound plate received
Compound 1 and one second agent each in 5 different doses centered
approximately on the EC.sub.50 of the given compound for a given
cell line, providing a total of 25 different dose combinations of
the two agents.
[0236] Compound arrays were distributed to assay plates using a 20
nl 384-well pin transfer manifold on a Janus MDT workstation
(Perkin Elmer). Each plate contained 8 replicates of all 5 by 5
compound concentrations in addition to five doses of each compound
on its own in quadruplicate. After addition of compounds, cell
plates were incubated for 5 days in a 37.degree. C. incubator. Cell
viability was evaluated using ATPlite (Perkin Elmer) following
manufacturer protocols. Data was analyzed using commercially
available CalcuSyn software and visualized using GraphPad Prism
Software. Isobolograms plotting each of the 25-dose combination of
Compound 1 and the second agents were generated and analyzed for
the presence of synergy. In the isobolograms, the straight line
connecting the abscissa and the ordinate values of 1.0 represents
growth inhibitions that were additive for the combination of the
two compounds. Plots that fall below the straight line represented
synergistic growth inhibitions, with plots that fall below that
line and one connecting the abscissa and the ordinate values of
0.75 represent mild synergy. Plots that fall between a line
connecting the abscissa and the ordinate values of 0.75 and a line
connecting the abscissa and the ordinate values of 0.25 represent
moderate synergy. Plots that fall below a line connecting the
abscissa and the ordinate values of 0.25 represent strong synergy.
Data points outside the maxima in each isobologram are indicated by
the number of asterisks at the top of the isobologram and represent
data points of no synergy.
[0237] The combined effect of Compound 1 and venetoclax was
examined on seven AML cell lines: THP1 (FIGS. 13A-13D), AML3 (FIGS.
14A-14C), HL60 (FIGS. 15A-15D), KG1 (FIG. 25A), ML-2 (FIG. 36),
KG-1 (FIG. 37), and OCI-M1 (not shown). Synergy was shown for this
combination in THP1, HL60, KG1, ML-2 and KG-1. OCI-M1 showed no
synergy and is an AML cell line that is known to have a high
expression of BCLXL.
[0238] We also examined the combined effect of Compound 1 and the
BET inhibitor JQ1 on four different AML cell lines: THP1 (FIGS.
9A-9D), AML3 (FIGS. 10A-10D), OCI-M1 (FIGS. 11A-11D) and HL60
(FIGS. 12A-12E). Synergy was observed for this combination in all
four AML cell lines tested.
[0239] We examined the combined effect of Compound 1 and the FLT3
inhibitor midostaurin on three different AML cell lines: THP1
(FIGS. 16A-16D), AML3 (FIGS. 17A-17D), and MV411 (FIGS. 18A-18D).
Synergy was observed for this combination in THP1 and MV411, while
AML3 showed a mostly additive effect.
[0240] We examined the combined effect of Compound 1 and the CDK9
inhibitor NVP2 on the Her2 amplified, ER.sup.-/PR.sup.- breast
cancer cell line AU565 (FIGS. 19A-19D). Synergy was observed for
the combination in this cell line.
[0241] We examined the combined effect of Compound 1 and the PARP
inhibitor niraparib on two different breast cancer cell
lines--HCC38--a TNBC cell line (FIGS. 20A-20E), and AU565 (FIGS.
21A-21E). Synergy was observed for this combination in both cell
lines.
[0242] In addition, we compared the ability of JQ1 alone, Compound
1 alone, and a combination of JQ1 and Compound 1 to modify
expression of various genes in two breast cancer cell lines. Two
triple-negative breast cancer cell lines (HCC70 and MDA-MB468) were
tested. The cell lines were treated with JQ1, Compound 1, the
JQ1/Compound 1 combination, or DMSO for four hours, after which
gene expression profiling via RNA-seq was performed. JQ1 was
administered to the cells at a 125 nM final concentration in both
single agent and combination experiments. Compound 1 was
administered to the cells at a 25 nM final concentration in both
single agent and combination experiments. Each experiment was
performed in triplicate. One replicate of MDA-MB-468 treated with
single-agent JQ1 was excluded from further analysis due to
quality.
[0243] We employed DESeq2 (Love et al., Genome Biology, 15(12):550,
2014) to identify changes in gene expression due to the JQ1,
Compound 1, and JQ1-Compound 1 combination treatment compared to
the control DMSO condition based on the RNA-seq data. Genes
resulting in a p-value below 0.01 and an absolute logFoldChange
above 0.5 were considered to be differentially expressed. In both
the HCC70 and MDA-MB468 cell-lines, the combination Compounds 1/JQ1
treatment produced more significantly downregulated genes than
single-agent Compound 1 or JQ1 treatments. We identified 3570
significantly downregulated genes from the combination JQ1/Compound
1 treatment in HCC70, while JQ1 alone resulted in 2874
downregulated genes and the individual Compound 1 treatment
resulted in 1782 downregulated genes. Similarly, 2414 genes were
downregulated in the combination treatment in the MDA-MB468
cell-line, while only 558 and 764 genes were downregulated given
the JQ1 and Compound 1 single agent treatments, respectively. A
large fraction of these significantly downregulated genes is unique
to the combination treatment. 798 of the 3570 downregulated genes
following combination treatment in the HCC70 cell-line were not
significantly downregulated in either of the individual treatment
conditions. The same is true for 1459 of 2414 genes in
MDA-MB468.
[0244] To identify the genes whose expression is impacted
synergistically by the drug combination, we modeled the effect of
each drug treatment on expression using a linear model. The
expression of each gene after the combination treatment can be
thought of as the combined impact of Compound 1, JQ1, and any
synergistic interaction between the drug treatments on baseline
gene expression. A linear model used to describe this relationship
could therefore be represented as:
Gene Expression after combination treatment=Baseline
Expression+Compound 1 effect+JQ1 effect+Combination effect
(Synergistic Impact)
[0245] By fitting the expression data for each gene using this
model, we were able to evaluate the effect of each treatment, and
identify cases where a synergistic interaction between Compound 1
and JQ1 impacts final gene expression. After fitting the linear
model to each gene, the weight and p-value of each term can be
evaluated. The combination term's weight signifies the mean change
resulting from a synergetic impact on expression, with a negative
weight indicating synergistic downregulation. Its associated
p-value represents the probability that the combination term is not
relevant to final gene expression. Using this approach, we
identified 1806 genes in HCC70 and 2205 genes in MDA-MB468 whose
expression change in the combination treatment was synergistic and
whose associated p-value was less than a threshold cutoff of 0.01.
The expression of these synergistic genes could not be explained by
an additive effect of Compound 1 and JQ1 alone. Several key
transcription factors implicated in breast cancer are downregulated
and demonstrate synergy under the combination treatment, including
GATA3 (Byrne et al., Histopathology, 2017), FOXC1 (Johnson et al.,
Oncotarget, 7(46):75729, 2016), and TGIF1 (Zhang et al., Cancer
Cell, 27(4):547-650, 2015).
[0246] The tables below show how each treatment affected expression
of the three genes.
TABLE-US-00006 TABLE 5 Effect of JQ1, Compound 1 or a Combination
Thereof of Expression of Certain Genes in Breast Cancer Cell Lines.
Average Average Expression Average Expression Average (TPM)
Expression (TPM) Expression Compound 1 + Gene Cell Line (TPM) DMSO
Compound 1 (TPM) JQ1 JQ1 GATA3 HCC70 32.685 22.404 29.574 13.098
MDA-MB468 34.244 21.773 32.491 11.237 FOXC1 HCC70 40.481 25.768
40.529 15.774 MDA-MB468 33.731 25.556 37.112 14.953 TGIF1 HCC70
62.523 68.293 52.509 30.868 MDA-MB468 145.88 134.08 151.60
65.215
TABLE-US-00007 TABLE 6 Linear Model Coefficients and Significance
of Synergy on Certain Genes for a Combination of Compound 1 and JQ1
in Breast Cancer Cell Lines. Synergy Synergy Coefficient Gene Cell
Line Coefficient Significance GATA3 HCC70 -6.1974 1.475 .times.
10.sup.-3 MDA-MB468 -8.7823 7.493 .times. 10.sup.-3 FOXC1 HCC70
-10.042 9.188 .times. 10.sup.-5 MDA-MB468 -13.985 3.317 .times.
10.sup.-3 TGIF1 HCC70 -27.411 7.978 .times. 10.sup.-6 MDA-MB468
-74.591 6.666 .times. 10.sup.-4
[0247] We also examined the combined effect of Compound 1 and three
different CDK4/6 inhibitors on the ER.sup.+ breast cancer cell line
T47D. Synergy was observed for all three CDK4/6 inhibitors in
combination with Compound 1 (palbociclib, FIGS. 26A-26C;
ribociclib, FIGS. 27A-27C; and abemaciclib, FIGS. 28A-28C).
[0248] We also examined the combined effect of Compound 1 and the
BET inhibitor JQ1 on Ewing's Sarcoma cell lines (SKES, RDES, A673)
as well as one osteosarcoma line (Saos2). Cells were grown to 70%
confluency in their media of preference based on the manufacturer's
recommendations. On the day of assay, cells were lifted and counted
using the Countess II FL (Life Technologies). Using a Biotek EL406,
50 .mu.L of preferred cell media containing 30,000 cells/ml was
distributed into black 384-well Nunc plates (Thermo) and allowed to
adhere overnight prior to compound addition. Compound arrays were
distributed to 384 well assay plates using Synergy Plate Format
with an HP D300e Digital Dispenser (HP). Compound 1 and JQ1 were
dissolved in DMSO to make a stock solution which allowed for
accurate dispensing. Compounds were plated in each quadrant of a
384 well plate in quadruplicate. Each quadrant contained test wells
with combination of Compound 1 and JQ1 as well as single agent
columns, and vehicle wells. After addition of compound, cell plates
were incubated for 3 days in a 37.degree. C. incubator. Cell
viability was evaluated using ATPlite (Perkin Elmer) following
manufacturer protocols. Data was analyzed in CalcuSyn utilizing the
median effect principle of presented by Chou-Talalay and visualized
using GraphPad Prism Software. Key parameters assessed were
combination index and dose reduction index.
[0249] Synergy was observed for JQ1 in combination with Compound 1
for all cell lines (SKES, FIG. 47; RDES, FIGS. 48A-48B; A673, FIGS.
49A-49B; Saos2, FIGS. 50A-50B).
Example 5. Inhibition of TNBC Cell Line Growth by Compound 1
Correlates with Low BCLXL Expression Levels
[0250] To determine the effect of Compound 1 on the growth of TNBC
cancer cells, four different human TNBC cell lines were
used--HCC70, MDA-MB-468, MDA-MB-453 and CAL120. Cells from each
cell line were plated separately at 50,000 cells/mL (100 uL per
well) in a black-walled 96 well plate and allowed to adhere
overnight. In parallel, cells were plated in a separate day 0 plate
to measure the number of cells present upon compound addition. The
next day, Compound 1 was added to the wells with an HP 300e
compound dispenser in a 10-point serial dilution and cells
incubated for 72 hours. On the same day Cell Titer Glo 2.0 reagent
was added to the day 0 plate, and the luminescence measured with an
Envision plate reader per manufacture protocol. After 72 hours Cell
Titer Glo 2.0 reagent was added to the plates and the luminescence
measured. GR curves were calculated as follows:
GR ( c ) = 2 log 2 ( x ( c ) / x 0 ) log 2 ( x ctrl / x 0 ) - 1 ,
##EQU00001##
wherein x(c) is the Compound 1 treatment luminescence); x.sub.0 is
the average Day 0 luminescence); and x.sub.ctrl is the average DMSO
treatment luminescence, and graphed using Graph Pad Prism. A GR
value of 1 indicates no growth inhibition; GR values between 0 and
1 indicate partial growth inhibition; a GR value of 0 indicates
cytostasis (no change from baseline); GR values less than 0
indicate cytotoxicity (cell number less than baseline); a GR value
of -1 indicates complete cell loss. FIG. 30A shows the results of
this experiment, with Compound 1 demonstrating almost complete
inhibition of growth of both HCC70 and MDA-MB-468 at concentrations
greater than 100 nM.
[0251] To determine the effect of Compound 1 on the expression of
BCL-XL, BCL-2, and MCL1, the same four triple negative breast
cancer (TNBC) cell lines (HCC70, MDA-MB-468, MDA-MB-453 and CAL120)
were seeded in a six-well plate at a density of 1.times.10.sup.6
cells/well and allowed to adhere overnight. Cells were then treated
with vehicle (DMSO), 50 nM of Compound 1, or 100 nM of Compound 1
for 24 hours. Cells were harvested, placed on ice, and resuspended
in RIPA lysis buffer supplemented with protease and phosphatase
inhibitors. Clarified lysates were prepared with 4.times.LSD
running buffer and boiled at 95.degree. C. for 5 minutes.
Equivalent amounts of sample (10 .mu.g total protein) were run on a
4-12% Bis-Tris gel and transferred to a PDVF membrane for Western
blotting using standard protocols. Membranes were blocked with
Licor TBS blocking buffer then probed with primary antibodies for
BCL-XL, BCL-2, MCL1, and beta-actin. Antibody-probed membranes were
visualized using an Odyssey imager. Sensitivity to Compound 1
correlated well with baseline BCLXL expression with HCC70 and
MDA-MB-468 showing the lowest levels of BCLXL expression (derived
by densitometry analysis of the Western blot shown in FIG. 29A and
shown graphically in FIG. 30B).
Example 6. Xenograft Models of TNBC and Ovarian Cancer
[0252] A. HCC70-Derived Xenografts
[0253] Subcutaneous HCC70 xenografts were established in BALC/c
nude mice at ChemPartner (Shanghai, China). Each mouse was
inoculated subcutaneously in the right flank with 5.times.10.sup.6
HCC70 cells (ATCC, CAT #: CRL-2315) in 0.2 ml of a 1:1 mixture of
base medium and Matrigel. Tumor sizes were measured in two
dimensions using a caliper, and the volumes were expressed in
mm.sup.3 using the formula: V=0.5 a.times.b.sup.2 where a and b are
the longest and shortest diameters of the tumor, respectively.
Compound 1 treatment was started when the average tumor size
reached 181 mm.sup.3.
[0254] Compound 1 was formulated in w/v 20% Captisol (pH 4-6) and
administered by i.v. twice weekly (BIW) at a final dose of 40 mg/kg
in a 10 ml/kg volume. Mice in the vehicle arm were given the same
dosing schedules, volumes, and formulations, but lacking Compound
1. Tumor volumes were measured twice weekly over the course of the
study. As shown in FIG. 31, treatment with Compound 1 caused a
reduction in tumor volume, while treatment with vehicle alone
resulted in an increase in tumor volume.
[0255] For the HCC70 xenograft mice, a sample of the tumor was
removed after a single dosing and prepared for Western blotting as
described in Example 2. As shown in FIG. 29B, treatment of the
HCC70 xenograft mouse with a single dose of 40 mg/kg of Compound 1
reduced MCL1 protein expression, confirming the results obtained
with this and other cell lines.
[0256] B. TNBC Patient-Derived Xenografts
[0257] Patient-derived xenograft (PDX) models from TNBC patients
(BR5010, BR5013, BR5015, and BR5023) were established in NOD-SCID
mice at Crown Bioscience (San Diego, USA). Cryo vials containing
tumor cells were thawed and prepared for injection into mice. Cells
were washed in PBS, counted, and resuspended in cold PBS at a
concentration of 50,000-100,000 viable cells/100 ul. Cell
suspensions were mixed with an equal volume of Cultrex ECM and kept
on ice during transport to the vivarium. Cells were prepared for
injections by withdrawing ECM-Cell mixture into a chilled slip-tip
syringe fitted with a 26G 7/8 (0.5 mm.times.22 mm) needle. The
filled syringes were kept on ice to avoid the solidification of
ECM. Each mouse was inoculated subcutaneously in the right flank
with 200 uL of the cell suspension. Tumor sizes were measured in
two dimensions using a caliper, and the volumes were expressed in
mm.sup.3 using the formula: V=0.5 a.times.b.sup.2, where a and b
are the longest and shortest diameters of the tumor, respectively.
Compound 1 treatment was started when the average tumor size
reached 150-200 mm.sup.3.
[0258] Compound 1 was formulated in w/v 20% Captisol (pH 4-6) and
administered by i.v. twice weekly (BIW) at a final dose of 40 mg/kg
or 30 mg/kg in a 10 ml/kg volume. Tumor volumes were measured twice
weekly over the course of the study. Growth of tumors in Compound 1
treated mice was compared to historical growth of untreated mice
for each model. The results of this experiment are shown in FIGS.
32A-32C. As can be seen in FIG. 32A, Compound 1 treatment of BR5010
xenografts consistently inhibited tumor growth, as compared to
untreated historical samples. However, Compound 1 had little
inhibitory effect on tumor growth in BR5013 (FIG. 32B), BR5015
(FIG. 32C) and BR5023 (FIG. 32D) xenografts.
[0259] To elucidate the cause of these different responses, we
looked at both BCL21 and CCNE1 mRNA levels in each of the
xenografts. Approximately 10-20 mg of snap-frozen xenograft tumor
tissue samples from PDX models BR5010, BR5013, and BR5015 were
pulverized using Cryoprep (Covaris CP02). A total of 750 uL of
Trizol reagent (Ambion 15596026) was added to the pulverized
sample. Total RNA was extracted using and RNA isolation kit
(Invitrogen AM1560), and concentrations of Total RNA were measured
with a Nano-drop microvolume spectrophotometer. RNA (100 ng) from
each sample was used as input for gene expression assay using the
Nanostring nCounter XT technology with the nCounter GX Human Cancer
Reference kit. Two independent tumors were assessed per model (T1,
T2). Higher CCNE1 expression (FIG. 33B) and lower BCL2L1 expression
(FIG. 33A) was observed in the responder line BR5010 compared to
the two non-responder lines.
[0260] To further evaluate the cause for high CCNE1 expression in
BR5010, we evaluated CCNE1 gene copy number in BR5010 and the
non-responder cell line BR5023. For DNA extraction, 10-20 mg of
pulverized PDX sample was suspended with 180 .mu.L ALT buffer and
20 .mu.L of proteinase K from DNeasy Blood & Tissue Kit (Qiagen
69504). Concentrations of total DNA were evaluated with a Nano-drop
microvolume spectrophotometer.
[0261] Whole-exome sequencing data from PDX models were analyzed by
WuXi NextCODE to determine mutations of interest in each sample and
mutations recurring in the cohort. All samples were analyzed using
the NextCODE Sequence Miner bioinformatics platform. Mouse read
filtering is performed by assessing sequence reads that are
"misaligned" to the human reference genome. Genes with the highest
number of variants were inspected and analyzed with NCBI BLAST
algorithms to confirm bona fide human reads. Germline variants were
filtered by removing all variants found in the single nucleotide
polymorphism database (dbSNP) with exception of those found in
COSMIC. Copy number variations were determined from the filtered
bam files using the CNVkit algorithm. Very high CCNE1 gene copy
number was observed in the responder line BR5010 whereas relatively
normal CCNE1 gene copy number was observed in non-responder line
BR5023 (FIG. 33C).
[0262] C. Ovarian Cancer Patient-Derived Xenografts
[0263] Patient-derived xenograft (PDX) models from ovarian
carcinoma patients (OV5387, OV14702, OV14972, OV15398, OV15576,
OV15696, OV15612, OV15631) were established in NOD-SCID mice at
Crown Bioscience (San Diego, USA). Cryo vials containing tumor
cells were thawed and prepared for injection into mice. Cells were
washed in PBS, counted, and resuspended in cold PBS at a
concentration of 50,000-100,000 viable cells/100 ul. Cell
suspensions were mixed with an equal volume of Cultrex ECM and kept
on ice during transport to the vivarium. Cells were prepared for
injections by withdrawing ECM-Cell mixture into a chilled slip-tip
syringe fitted with a 26G 7/8 (0.5 mm.times.22 mm) needle. The
filled syringes were kept on ice to avoid the solidification of
ECM. Each mouse was inoculated subcutaneously in the right flank
with 200 uL of the cell suspension. Tumor sizes were measured in
two dimensions using a caliper, and the volumes were expressed in
mm3 using the formula: V=0.5 a.times.b.sup.2 where a and b are the
longest and shortest diameters of the tumor, respectively. Compound
1 treatment was started when the average tumor size reached 150-200
mm.sup.3.
[0264] Compound 1 was formulated in w/v 20% Captisol (pH 4-6) and
administered by i.v. twice weekly (BIW) at a final dose of 40 mg/kg
or 30 mg/kg in a 10 ml/kg volume. Tumor volumes were measured twice
weekly over the course of the study. Growth of tumors in Compound
1-treated mice was compared to historical growth of untreated mice
for each model. As can be seen from FIGS. 34A-34H, four of the
xenografts responded to treatment (FIGS. 34A-34D), while four did
not (FIGS. 34E-34H).
[0265] In order to elucidate the cause of these different
responses, we looked at the level of various proteins from the
xenografts. Tumor tissue collected from each of the patient
xenografts and was prepared for and subject to Western blot
analysis as described in Example 2 using primary antibodies against
RB1 (Cell Signaling, CST9309), CCNE1 (Santa Cruz, sc-247), FGFR1
(Cell Signaling, CST9740) or .beta.-ACTIN. For some models, two
independent tumors were assessed (T1, T2). As shown in FIG. 35, all
responders demonstrated either low RB1 expression or high CCNE1
expression. All 4 non-responders had relatively high RB1
expression; 1/4 non-responders had increased CCNE1 expression
(OV15696), although expression levels were lower than those
observed in the CCNE1.sup.HI responsive model OV15612.
[0266] We then preformed RNA extraction to determine gene
expression and, DNA extraction to determine gene copy number and
mutations, and H3K27Ac ChIP-Seq to determine super enhancers for
the various PDX tumors. For RNA extraction, PDX tumors were
pulverized using Cryoprep (Covaris CP02). Ten to twenty mg of the
pulverized sample were suspended with 750 .mu.L of Trizol reagent
(Ambion 15596026). Total RNA was extracted using RNA isolation kit
(Invitrogen AM1560). For DNA extraction, 10-20 mg of pulverized PDX
sample were suspended with 180 .mu.L ALT buffer and 20 .mu.L of
proteinase K from DNeasy Blood & Tissue Kit (Qiagen 69504).
Concentrations of total RNA and DNA were evaluated with a Nano-drop
microvolume spectrophotometer. One-hundred ng of RNA from each
sample were used as input for gene expression assay using
Nanostring nCounter XT technology (nCounter GX Human Pan Cancer kit
with 30 Custom PLUS gene set). One .mu.g of DNA was sent to
WuXiNextCODE for Whole Exome Sequencing (WES-Agilent-V6-100x) for
analysis of gene copy number and mutations (single nucleotide
variants, InDels).
[0267] Samples were analyzed using the NextCODE Sequence Miner
bioinformatics platform. Mouse read filtering was performed by
assessing sequence reads that were misaligned to the human
reference genome. Genes with the highest number of variants were
inspected and analyzed with NCBI BLAST algorithms to confirm bona
fide human reads. Germline variants were filtered by removing all
variants found in the single nucleotide polymorphism database
(dbSNP) with exception of those found in COSMIC. Copy number
variations were determined from the filtered bam files using the
CNVkit algorithm.
[0268] For H3K27Ac ChIP-Seq, 10-50 mg of pulverized PDX samples
were cross-linked with 1% formaldehyde in PBS for 8 minutes,
cross-linking process were quenched by adding 2.5M Glycine. Tissues
were then lysed with Lysis buffer LB1 (Boston Bioproducts, CHP-126)
and LB2 (Boston Bioproducts, CHP-127) for 10 minutes sequentially.
Lysed samples were sonicated using focused ultrasonicator (Covaris,
E220), H3k27Ac antibody-conjugated magnetic beads (Abcam ab4729,
Invitrogen, 10004D) were added after sonication and incubated
overnight in 4.degree. C. Samples were washed and eluted off the
beads with Elution buffer (Boston Bioproducts, CHP-153). Reverse
cross-link was performed by incubating samples in 65.degree. C.
overnight. DNA was precipitated and cleaned using phenol chloroform
extraction (Sigma, P3803). Eluted DNA was sequenced and
super-enhancer analysis was performed based on the SE-scoring
algorithm of M. R. McKeown et al., Cancer Discov, 2017, 7(10), pp.
1136-1153.
[0269] One of the ovarian PDX models that responded to Compound 1
(0V15612) contained a strong super enhancer associated with the
FGFR1 gene (FIG. 45A), and highly overexpresses FGFR1, CDK6, and
CCND2 mRNA (FIGS. 45B-45D), and FGFR1 protein (FIG. 45E) as
compared to other ovarian PDX models we tested. Another ovarian PDX
model that responded to Compound 1 (OV15398) had very low
expression of the tumor suppressor CDKN2A (P16), a potent inhibitor
of CDK4/6 activity compared to other ovarian PDX models (FIG. 46).
Yet another ovarian PDX that responded to Compound 1 (OV14702)
contained a single copy of the RB1 gene, which had a frameshift
mutation (pE204X) indicating it is RB1 null. These results confirm
that Compound 1 is effective in cancers that are characterized by
molecular alterations that confer CDK 4/6 inhibitor resistance.
Example 7. A Combination of Venetoclax and Compound 1 Act
Synergistically on a BCLXL.sup.LO AML Cell Line (KG-1)
Xenograft
[0270] Subcutaneous KG-1 xenografts were established in CB17 SCID
mice at ChemPartner (Shanghai, China). Each mouse was inoculated
subcutaneously in the right flank with 5.times.10.sup.6 KG-1 cells
(ATCC, CAT #: CCL-246) in 0.2 ml of a 1:1 mixture of base medium
and Matrigel. Tumor sizes were measured in two dimensions using a
caliper, and the volumes were expressed in mm3 using the formula:
V=0.5 a.times.b.sup.2 where a and b are the longest and shortest
diameters of the tumor, respectively. Compound 1 and/or venetoclax
treatments were started when the average tumor size reached 200
mm.sup.3.
[0271] Compound 1 was formulated in 20% w/v Captisol (pH 4-6) and
administered by i.v. once weekly (QW) at a final dose of 40 mg/kg
in a 10 ml/kg volume. Venetoclax was formulated in 60% Phosal 50
propylene glycol (PG), 30% polyethylene glycol (PEG 400), 10%
ethanol, and administered by oral gavage once daily (QD) at a final
dose of 50 mg/kg in a 10 ml/kg volume. Mice in the combination arm
were given the same dosing schedules, volumes, and formulations for
each agent. Mice in the vehicle arm were given the same dosing
schedules, volumes, and formulations, but lacking Compound 1 and
venetoclax. Tumor volumes were measured twice weekly over the
course of the study.
[0272] The results of this experiment are shown in FIG. 38, where
the combination of venetoclax and Compound 1 had greater inhibitory
effect on tumor growth that either agent administered alone.
Example 8. Combinations of Compound 1 with Carboplatin,
Oxaliplatin, Olaparib, or Paclitaxel Act Synergistically on Ovarian
Cancer Cell Lines
[0273] Ovarian cell lines (OvCar3, CaOV3, COV644, COV318,
Kuramochi, OV-90, SKOV3, TOV21G, A2780, ES2, COV504, COV362) were
grown to 70% confluency in their media of preference based on the
manufacturer recommendations. On the day of assay, cells were
lifted, and counted using the Countess II FL (Life Technologies).
Using a Biotek EL406, 50 .mu.L of preferred cell media containing
30,000 cells/ml was distributed into black 384-well Nunc plates
(Thermo) and allowed to adhere overnight prior to compound
addition. Compound arrays were distributed to 384 well assay plates
using Synergy Plate Format with an HP D300e Digital Dispenser (HP).
Compound 1 and other test agents were dissolved in DMSO to make a
stock solution which allowed for accurate dispensing. However, due
to solubility and reactivity, the platinum agents carboplatin and
oxaliplatin were dissolved in water with addition of 0.03% Tween-20
to allow for dispensing with digital printer. Compounds were plated
in each quadrant of a 384 well plate in quadruplicate. Each
quadrant contained test wells with combination of Compound 1 and
test agent as well as single agent columns, and vehicle wells.
[0274] After addition of compound, cell plates were incubated for 3
days in a 37.degree. C. incubator. Cell viability was evaluated
using ATPlite (Perkin Elmer) following manufacturer protocols. Data
was analyzed in CalcuSyn utilizing the median effect principle of
presented by Chou-Talalay and visualized using GraphPad Prism
Software. Key parameters assessed were combination index and dose
reduction index.
[0275] The results of these studies are shown in isobolograms in
FIG. 39A (carboplatin), FIGS. 40A-40B (oxaliplatin), FIGS. 41A-41B
(olaparib) and FIGS. 42A-42B (paclitaxel). For each agent, synergy
is seen in the majority of ovarian cancer cell lines tested. The
A2780 cell line is known to be resistant to platinum-based agents,
as shown in FIG. 39B. Data from this cell line, combined with the
demonstration that Compound 1 causes a decrease in mRNA levels of
several genes involved in DNA damage repair (Example 9) and
therefore, resistance to platinum-based agent, supports the use of
CDK7 inhibitors (e.g., Compound 1) to overcome such resistance.
Example 9. Compound 1 Causes a Downregulation in mRNA Expression
from Genes Involved in DNA Repair in AML, Breast Cancer, and
Ovarian Cancer Cell Lines
[0276] THP1 is an AML cell line. THP1 cells (1.times.10.sup.6/well)
were plated in 6-well plates and treated with vehicle (DMSO), 100
nM Compound 1, 25 nM NVP2 (a CDK9 inhibitor), 250 nM JQ1 (a BRD4
inhibitor) or 200 nM Flavopiridol (a pan-CDK inhibitor) for 6 hrs,
after which cells were harvested and total RNA (1,000 ng) isolated.
RNA levels of the DNA damage repair genes Rad51, CHEK1 and CHEK2
were analyzed by microarray. Experiments were done in triplicates.
We performed RMA normalization of the data using the "affy" package
from Bioconductor. The command used is shown below and includes
background correction, normalization and summarization:
TABLE-US-00008 rma_Result <-expresso(raw_data,
bgcorrect.method="rma",normalize=TRUE,pmcorrect.method="pmonly",
summary.method="medianpolish")
Next, we performed loess normalization using only spike-ins to
enable comparison of expression values across multiple samples. The
command used was:
TABLE-US-00009 ma_expr_norm <-normalize.loess(exprs(rma_Result),
subset=grep("ERCC-",rownames(exprs(rma_Result))))
The results are shown in FIG. 43.
[0277] Breast cancer cell lines MDA-MB-468, MDA-MB-231, Ca1120 and
MDA-MB-453 were plated at 200,000 cells/well in a 6-well plate the
day before treatment. The next day the cells were treated with
vehicle, 50 nM Compound 1 or 50 nM Paclitaxel for 6 hrs, after
which cells were harvested and total RNA collected using RNeasy
mini kit (Qiagen). Total RNA (500 ng) was reverse transcribed using
Quantitect Reverse Transcription kit (Qiagen). Quantitative PCR for
each of Rad51, CHEK1 and CHEK2 was performed on QuantStudio 6 Flex
(Applied Biosystems) using Power SYBR green PCR master mix (Thermo
Fisher Scientific) and primers specific for each of those genes.
The change in gene expression, relative to housekeeping gene RPL27,
was quantified using DDCt method. The results are shown in FIG.
44.
[0278] Ovarian Cell lines OvCar3, TOV21G, A2780 and COV318 were
plated at 500,000 cells/well in a 6-well plate the day before
treatment. The next day the cells were treated with vehicle or 50
nM Compound 1 for 0, 6, or 16 hours after which cells were
harvested and total RNA collected using RNeasy mini kit (Qiagen).
Changes in mRNA between samples were analyzed with Nanostring.TM.
PanCancer Pathways Panels specifically analyzing genes related to
homologous recombination deficiency and carboplatin sensitivity
(ie. BRCA1, BRCA2, Rad51, ATM, ATR, MSH2, MSH6). Nanostring signal
intensities were first normalized to housekeeping genes across all
cell lines, and then normalized to the 0 hour timepoint within a
cell line. All genes (except Rad51) were downregulated at 16 hours
across all cell lines (A2780, FIG. 51; COV318, FIG. 52; TOV21G,
FIG. 53; OvCar3, FIG. 54).
Example 10: Compound 1 Enhances Carboplatin Tumor Growth Inhibition
in Ovarian Cancer Xenografts
[0279] Subcutaneous TOV21G xenografts were established in BALB/c
nude mice at ChemPartner (Shanghai, China). Each mouse was
inoculated subcutaneously in the right flank with 5.times.10.sup.6
TOV-21G cells (Human ovarian cancer, ATCC, CRL-11730, 5034683) in
0.2 ml of a 1:1 mixture of base medium and Matrigel. Tumor sizes
were measured in two dimensions using a caliper, and the volumes
were expressed in mm3 using the formula: V=0.5 a.times.b.sup.2
where a and b are the longest and shortest diameters of the tumor,
respectively. Compound 1 and/or carboplatin treatments were started
when the average tumor size reached 150 mm.sup.3.
[0280] Subcutaneous OVCAR3 xenografts were established in BALB/c
nude mice at Crown Bioscience Inc. (Beijing, China). Each mouse was
inoculated subcutaneously in the right flank with 1.times.10.sup.7
OVCAR-3 cells (Human ovarian cancer, ATCC HTH-161, NIH:OVCAR-3) in
0.1 ml of a 1:1 mixture of base medium and Matrigel. Tumor sizes
were measured in two dimensions using a caliper, and the volumes
were expressed in mm3 using the formula: V=0.5 a.times.b.sup.2
where a and b are the longest and shortest diameters of the tumor,
respectively. Compound 1 and/or carboplatin treatments were started
when the average tumor size reached 150 mm.sup.3.
[0281] Subcutaneous A2780 xenografts were established in BALB/c
nude mice at Crown Bioscience Inc. (Beijing, China). Each mouse was
inoculated subcutaneously in the right flank with 1.times.10.sup.7
A2780 cells (Human ovarian cancer, ECACC 93112519, A2780) in 0.1 ml
of a 1:1 mixture of base medium and Matrigel. Tumor sizes were
measured in two dimensions using a caliper, and the volumes were
expressed in mm3 using the formula: V=0.5 a.times.b.sup.2 where a
and b are the longest and shortest diameters of the tumor,
respectively. Compound 1 and/or carboplatin treatments were started
when the average tumor size reached 270 mm.sup.3.
[0282] Carboplatin was formulated in water and administered by i.p.
once weekly (QW) at a final dose of 50 mg/kg in a 10 ml/kg volume.
Compound 1 was formulated in 20% w/v Captisol (pH 4-6) and
administered by i.v. once weekly (QW) at a final dose of 20 mg/kg
in a 10 ml/kg volume for A2780, and a final dose of 30 mg/kg in a
10 ml/kg volume for TOV21G and OVCAR3. SY-1365 was administered 8
hours after carboplatin for each model. Mice in the combination arm
were given the same dosing schedules, volumes, and formulations for
each agent. Mice in the vehicle arm were given the same dosing
schedules, volumes, and formulations, but lacking Compound 1 and
carboplatin. Tumor volumes were measured twice weekly over the
course of the study.
[0283] The results of these experiments show that the combination
of carboplatin and Compound 1 had greater inhibitory effect on
tumor growth than either agent administered alone in all three
xenograft models (TOV21G, FIG. 55; OVCAR3, FIG. 56; A2780, FIG.
57).
[0284] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process. In this application, unless otherwise clear from
context, (i) the term "a" may be understood to mean "at least one";
(ii) the term "or" may be understood to mean "and/or"; (iii) the
terms "comprising" and "including" may be understood to encompass
itemized components or steps whether presented by themselves or
together with one or more additional components or steps; and (iv)
the terms "and (v) where ranges are provided, endpoints are
included.
[0285] Furthermore, the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims are introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, every possible subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It should it be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements and/or features, certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements and/or features. For purposes of
simplicity, those embodiments have not been specifically set forth
in haec verba herein. Where ranges are given, endpoints are
included. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or sub-range within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0286] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, that there
are many equivalents to the specific embodiments of the disclosure
described and claimed herein. Such equivalents are intended to be
encompassed by the following claims.
* * * * *